Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof

ABSTRACT

The present invention relates to new immunogenic compositions comprising conjugated  Streptococcus pneumoniae  capsular saccharide antigens (glycoconjugates) and uses thereof. Immunogenic compositions of the present invention will typically comprise at least one glycoconjugate from a  S. pneumoniae  serotype not found in PREVNAR®, SYNFLORIX® and/or PREVNAR 13®. The invention also relates to vaccination of human subjects, in particular infants and elderly, against pneumoccocal infections using said novel immunogenic compositions.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.14/597,488, filed Jan. 15, 2015, now issued as U.S. Pat. No. 9,492,559,which claims the priority benefit of U.S. Provisional Application No.61/929,547, filed Jan. 21, 2014, the entireties of which are herebyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to new immunogenic compositions comprisingconjugated capsular saccharide antigens (glycoconjugates) and usesthereof. Immunogenic compositions of the present invention willtypically comprise glycoconjugates, wherein the saccharides are derivedfrom serotypes of Streptococcus pneumoniae. The invention also relatesto vaccination of human subjects, in particular infants and elderly,against pneumoccocal infections using said novel immunogeniccompositions.

BACKGROUND OF THE INVENTION

Infections caused by pneumococci are a major cause of morbidity andmortality all over the world. Pneumonia, febrile bacteraemia andmeningitis are the most common manifestations of invasive pneumococcaldisease, whereas bacterial spread within the respiratory tract mayresult in middle-ear infection, sinusitis or recurrent bronchitis.Compared with invasive disease, the non-invasive manifestations areusually less severe, but considerably more common.

In Europe and the United States, pneumococcal pneumonia is the mostcommon community-acquired bacterial pneumonia, estimated to affectapproximately 100 per 100,000 adults each year. The correspondingfigures for febrile bacteraemia and meningitis are 15-19 per 100 000 and1-2 per 100,000, respectively. The risk for one or more of thesemanifestations is much higher in infants and elderly people, as well asimmune compromised persons of any age. Even in economically developedregions, invasive pneumococcal disease carries high mortality; foradults with pneumococcal pneumonia the mortality rate averages 10%-20%,whilst it may exceed 50% in the high-risk groups. Pneumonia is by farthe most common cause of pneumococcal death worldwide.

The etiological agent of pneumococcal diseases, Streptococcus pneumoniae(pneumococcus), is a Gram-positive encapsulated coccus, surrounded by apolysaccharide capsule. Differences in the composition of this capsulepermit serological differentiation between about 91 capsular types, someof which are frequently associated with pneumococcal disease, othersrarely. Invasive pneumococcal infections include pneumonia, meningitisand febrile bacteremia; among the common non-invasive manifestations areotitis media, sinusitis and bronchitis.

Pneumococcal conjugate vaccines (PCVs) are pneumococcal vaccines used toprotect against disease caused by S. pneumoniae (pneumococcus). Thereare currently three PCV vaccines available on the global market:PREVNAR® (called PREVENAR® in some countries) (heptavalent vaccine),SYNFLORIX® (a decavalent vaccine) and PREVNAR 13® (tridecavalentvaccine).

The recent development of widespread microbial resistance to essentialantibiotics and the increasing number of immunocompromised personsunderline the need for pneumococcal vaccines with even broaderprotection.

In particular, there is a need to address remaining unmet medical needfor coverage of pneumococcal disease due to serotypes not found inPREVNAR 13® and potential for serotype replacement over time. Thespecific serotypes causing disease beyond the 13 in PREVNAR 13® vary byregion, population, and may change over time due to acquisition ofantibiotic resistance, pneumococcal vaccine introduction and seculartrends of unknown origin. There is a need for immunogenic compositionsthat can be used to induce an immune response against additionalStreptococcus pneumoniae serotypes in humans and in particular inchildren less than 2 years old.

An object of the new immunogenic compositions of the present inventionis to provide for appropriate protection against S. pneumoniae serotypesnot found in PREVNAR 13®. In one aspect, an object of the immunogeniccompositions of the present invention is to provide for appropriateprotection against S. pneumoniae serotypes not found in PREVNAR®(heptavalent vaccine), SYNFLORIX® and/or PREVNAR 13® while maintainingan immune response against serotypes currently covered by said vaccines.

SUMMARY OF THE INVENTION

The present invention relates to an immunogenic composition comprisingat least one glycoconjugate selected from the group consisting of aglycoconjugate from S. pneumoniae serotype 15B, a glycoconjugate from S.pneumoniae serotype 22F, a glycoconjugate from S. pneumoniae serotype33F, a glycoconjugate from S. pneumoniae serotype 12F, a glycoconjugatefrom S. pneumoniae serotype 10A, a glycoconjugate from S. pneumoniaeserotype 11A and a glycoconjugate from S. pneumoniae serotype 8.

In one aspect, the invention provides an immunogenic compositioncomprising at least one glycoconjugate from S. pneumoniae serotype 15B,at least one glycoconjugate from S. pneumoniae serotype 22F and at leastone glycoconjugate from S. pneumoniae serotype 33F.

In another aspect the invention provides an immunogenic compositioncomprising at least one glycoconjugate from S. pneumoniae serotype 15B,at least one glycoconjugate from S. pneumoniae serotype 22F, at leastone glycoconjugate from S. pneumoniae serotype 33F, at least oneglycoconjugate from S. pneumoniae serotype 12F, at least oneglycoconjugate from S. pneumoniae serotype 10A, at least oneglycoconjugate from S. pneumoniae serotype 11A and at least oneglycoconjugate from S. pneumoniae serotype 8.

In an aspect the above immunogenic composition further comprisesglycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and23F.

In another aspect the above immunogenic composition further comprisesglycoconjugates from S. pneumoniae serotypes 1, 5 and 7F.

In another aspect the above immunogenic composition further comprisesglycoconjugates from S. pneumoniae serotypes 6A and 19A.

In another aspect the above immunogenic composition further comprisesglycoconjugates from S. pneumoniae serotype 3.

In another aspect the above immunogenic composition further comprisesglycoconjugates from S. pneumoniae serotype 2, 9N, 17F, 20 and/or 15C.

In an aspect the above immunogenic composition does not comprisecapsular saccharide from S. pneumoniae serotype 9N, 9A and/or 9L.

In an aspect the above immunogenic composition is a 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20-valent pneumococcal conjugatecomposition. In an aspect the above immunogenic composition is a 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or25-valent pneumococcal conjugate composition.

In an aspect the glycoconjugates are individually conjugated to acarrier protein selected form the group consisting of DT (Diphtheriatoxin), TT (tetanus toxoid), CRM₁₉₇, other DT mutants, PD (Haemophilusinfluenzae protein D), or immunologically functional equivalentsthereof.

In one aspect, the invention provides a container filled with any of theimmunogenic composition defined in the present document.

In one aspect, the invention provides any of the immunogenic compositiondefined in the present document for use as a medicament, in particularfor use as a vaccine.

In one aspect, the invention provides a method of preventing, treatingor ameliorating an infection, disease or condition associated with S.pneumoniae in a subject, comprising administering to the subject animmunologically effective amount of any of the immunogenic compositiondefined in the present document.

FIGURES

FIG. 1 shows a repeating polysaccharide structure of S. pneumoniaeserotype 8 (Pn-8) capsular polysaccharide.

FIG. 2 shows a repeating polysaccharide structure of S. pneumoniaeserotype 10A (Pn-10A) capsular polysaccharide.

FIG. 3 shows a repeating polysaccharide structure of S. pneumoniaeserotype 11A (Pn-11A) capsular polysaccharide.

FIG. 4 shows a repeating polysaccharide structure of S. pneumoniaeserotype 12F (Pn-12F) capsular polysaccharide.

FIG. 5 shows a repeating polysaccharide structure of S. pneumoniaeserotype 15B (Pn-15B) capsular polysaccharide.

FIG. 6 shows a repeating polysaccharide structure of S. pneumoniaeserotype 22F (Pn-22F) capsular polysaccharide.

FIG. 7 shows a repeating polysaccharide structure of S. pneumoniaeserotype 33F (Pn-33F) capsular polysaccharide.

FIG. 8 shows a representative process flow diagram for the activation(A) and conjugation (B) processes which can be used in the preparationof Pn-33F glycoconjugate.

FIG. 9 shows the effect on DO by varying amount of NCS in the TEMPO/NCSoxidation reaction.

FIG. 10 shows evaluation of Pn-12F glycoconjugates stability.

FIG. 11 Cross-Functional OPA Responses. A subset of 59 sera from adultsvaccinated with a 13 valent Pneumococcal Conjugate Vaccine (US Study6115A1-004; ClinicalTrials.gov Identifier: NCT00427895) was assessed inOPAs for the presence of functional antibodies against serotypes 9V, 9A,9L, and 9N. The percent of samples with OPA positive titer (i.e., ≥1:8)is indicated above each group. Geometric mean titers (GMT) are listed inthe x axis below each group.

FIG. 12 Cross-Functional OPA Responses of Sixty-six Matched pre/postSera. A subset of 66 matched pre- and post-vaccinated serum panel fromadults vaccinated with a 13 valent Pneumococcal Conjugate Vaccine (study6115A1-3005; ClinicalTrials.gov Identifier: NCT00546572) were assessedin OPAs for the presence of functional antibodies against serotypes 9V,9A, 9L, and 9N. The percent of samples with OPA positive titer (i.e.,≥1:8) is indicated above each group. Geometric mean titers (GMT) arelisted in the x axis below each group.

FIG. 13 Reverse cumulative distribution curves (RCDC) of pre and postImmunization—pneumococcal serotype 9V (Pn9V).

Reverse cumulative distribution curves of OPA titers to serotype 9V froma matched pre- and post-vaccination serum panel (N=66) vaccinated with a13 valent Pneumococcal Conjugate Vaccine (study 6115A1-3005;ClinicalTrials.gov Identifier: NCT00546572). The plots represent thepercent of sera with OPA positive titer (i.e., ≥1:8).

FIG. 14 Reverse cumulative distribution curves (RCDC) of pre and postImmunization—pneumococcal serotype 9A (Pn9A).

Reverse cumulative distribution curves of OPA titers to serotype 9A froma matched pre- and post-vaccination serum panel (N=66) vaccinated with a13 valent Pneumococcal Conjugate Vaccine (study 6115A1-3005;ClinicalTrials.gov Identifier: NCT00546572). The plots represent thepercent of sera with OPA positive titer (i.e., ≥1:8).

FIG. 15 Reverse cumulative distribution curves (RCDC) of pre and postImmunization—pneumococcal serotype 9L (Pn9L).

Reverse cumulative distribution curves of OPA titers to serotype 9L froma matched pre- and post-vaccination serum panel (N=66) vaccinated with a13 valent Pneumococcal Conjugate Vaccine (study 6115A1-3005;ClinicalTrials.gov Identifier: NCT00546572). The plots represent thepercent of sera with OPA positive titer (i.e., ≥1:8).

FIG. 16 Reverse cumulative distribution curves (RCDC) of pre and postImmunization—pneumococcal serotype 9N (Pn9N).

Reverse cumulative distribution curves of OPA titers to serotype 9N froma matched pre- and post-vaccination serum panel (N=66) vaccinated with a13 valent Pneumococcal Conjugate Vaccine (study 6115A1-3005;ClinicalTrials.gov Identifier: NCT00546572). The plots represent thepercent of sera with OPA positive titer (i.e., ≥1:8).

1 Immunogenic Compositions of the Invention

Immunogenic compositions of the present invention will typicallycomprise conjugated capsular saccharide antigens (also namedglycoconjugates), wherein the saccharides are derived from serotypes ofS. pneumoniae.

Preferably, the number of S. pneumoniae capsular saccharides can rangefrom 8 different serotypes (or “v”, valences) to 20 different serotypes(20 v). In one embodiment there are 8 different serotypes. In oneembodiment there are 9 different serotypes. In one embodiment there are10 different serotypes. In one embodiment there are 11 differentserotypes. In one embodiment there are 12 different serotypes. In oneembodiment there are 13 different serotypes. In one embodiment there are14 different serotypes. In one embodiment there are 15 differentserotypes. In one embodiment there are 16 different serotypes. In anembodiment there are 17 different serotypes. In an embodiment there are18 different serotypes. In an embodiment there are 19 differentserotypes. In an embodiment there are 20 different serotypes. Thecapsular saccharides are conjugated to a carrier protein to formglycoconjugates as described here below.

If the protein carrier is the same for 2 or more saccharides in thecomposition, the saccharides could be conjugated to the same molecule ofthe protein carrier (carrier molecules having 2 or more differentsaccharides conjugated to it) [see for instance WO2004/083251].

In a preferred embodiment though, the saccharides are each individuallyconjugated to different molecules of the protein carrier (each moleculeof protein carrier only having one type of saccharide conjugated to it).In said embodiment, the capsular saccharides are said to be individuallyconjugated to the carrier protein.

For the purposes of the invention the term ‘glycoconjugate’ indicates acapsular saccharide linked covalently to a carrier protein. In oneembodiment a capsular saccharide is linked directly to a carrierprotein. In a second embodiment a bacterial saccharide is linked to aprotein through a spacer/linker.

1.1 Carrier Protein of the Invention

A component of the glycoconjugate of the invention is a carrier proteinto which the saccharide is conjugated. The terms “protein carrier” or“carrier protein” or “carrier” may be used interchangeably herein.Carrier proteins should be amenable to standard conjugation procedures.

In a preferred embodiment, the carrier protein of the glycoconjugates isselected in the group consisting of: DT (Diphtheria toxin), TT (tetanustoxoid) or fragment C of TT, CRM₁₉₇ (a nontoxic but antigenicallyidentical variant of diphtheria toxin), other DT mutants (such asCRM176, CRM228, CRM45 (Uchida et al. (1973) J. Biol. Chem.218:3838-3844), CRM9, CRM102, CRM103 or CRM107; and other mutationsdescribed by Nicholls and Youle in Genetically Engineered Toxins, Ed:Frankel, Marcel Dekker Inc. (1992); deletion or mutation of Glu-148 toAsp, Gln or Ser and/or Ala 158 to Gly and other mutations disclosed inU.S. Pat. Nos. 4,709,017 and 4,950,740; mutation of at least one or moreresidues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutationsdisclosed in U.S. Pat. Nos. 5,917,017 and 6,455,673; or fragmentdisclosed in U.S. Pat. No. 5,843,711, pneumococcal pneumolysin (ply)(Kuo et al. (1995) Infect Immun 63:2706-2713) including ply detoxifiedin some fashion, for example dPLY-GMBS (WO 2004/081515, WO 2006/032499)or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (sequences ofPhtA, PhtB, PhtD or PhtE are disclosed in WO 00/37105 and WO 00/39299)and fusions of Pht proteins, for example PhtDE fusions, PhtBE fusions,Pht A-E (WO 01/98334, WO 03/054007, WO 2009/000826), OMPC (meningococcalouter membrane protein), which is usually extracted from Neisseriameningitidis serogroup B (EP0372501), PorB (from N. meningitidis), PD(Haemophilus influenzae protein D; see, e.g., EP0594610 B), orimmunologically functional equivalents thereof, synthetic peptides(EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO 94/03208),pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines,growth factors or hormones (WO 91/01146), artificial proteins comprisingmultiple human CD4+ T cell epitopes from various pathogen derivedantigens (Falugi et al. (2001) Eur J Immunol 31:3816-3824) such as N19protein (Baraldoi et al. (2004) Infect Immun 72:4884-4887) pneumococcalsurface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337),toxin A or B of Clostridium difficile (WO 00/61761), transferrin bindingproteins, pneumococcal adhesion protein (PsaA), recombinant Pseudomonasaeruginosa exotoxin A (in particular non-toxic mutants thereof (such asexotoxin A bearing a substitution at glutamic acid 553 (Douglas et al.(1987) J. Bacteriol. 169(11):4967-4971)). Other proteins, such asovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA)or purified protein derivative of tuberculin (PPD) also can be used ascarrier proteins. Other suitable carrier proteins include inactivatedbacterial toxins such as cholera toxoid (e.g., as described in WO2004/083251), Escherichia coli LT, E. coli ST, and exotoxin A from P.aeruginosa.

In a preferred embodiment, the carrier protein of the glycoconjugates isindependently selected from the group consisting of TT, DT, DT mutants(such as CRM₁₉₇), H. influenzae protein D, PhtX, PhtD, PhtDE fusions(particularly those described in WO 01/98334 and WO 03/054007),detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B ofC. difficile and PsaA.

In an embodiment, the carrier protein of the glycoconjugates of theinvention is DT (Diphtheria toxoid). In another embodiment, the carrierprotein of the glycoconjugates of the invention is TT (tetanus toxoid).

In another embodiment, the carrier protein of the glycoconjugates of theinvention is PD (H. influenzae protein D; see, e.g., EP0594610 B).

In a preferred embodiment, the capsular saccharides of the invention areconjugated to CRM₁₉₇ protein. The CRM₁₉₇ protein is a nontoxic form ofdiphtheria toxin but is immunologically indistinguishable from thediphtheria toxin. CRM₁₉₇ is produced by Corynebacterium diphtheriaeinfected by the nontoxigenic phage β197^(tox−) created bynitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchidaet al. (1971) Nature New Biology 233:8-11). The CRM₁₉₇ protein has thesame molecular weight as the diphtheria toxin but differs therefrom by asingle base change (guanine to adenine) in the structural gene. Thissingle base change causes an amino acid substitution (glutamic acid forglycine) in the mature protein and eliminates the toxic properties ofdiphtheria toxin. The CRM₁₉₇ protein is a safe and effective T-celldependent carrier for saccharides. Further details about CRM₁₉₇ andproduction thereof can be found, e.g., in U.S. Pat. No. 5,614,382.

In an embodiment, the capsular saccharides of the invention areconjugated to CRM₁₉₇ protein or the A chain of CRM₁₉₇ (see CN103495161).In an embodiment, the capsular saccharides of the invention areconjugated the A chain of CRM₁₉₇ obtained via expression by geneticallyrecombinant E. coli (see CN103495161). In an embodiment, the capsularsaccharides of the invention are all conjugated to CRM₁₉₇. In anembodiment, the capsular saccharides of the invention are all conjugatedto the A chain of CRM₁₉₇.

Accordingly, in frequent embodiments, the glycoconjugates of theinvention comprise CRM₁₉₇ as the carrier protein, wherein the capsularpolysaccharide is covalently linked to CRM₁₉₇.

1.2 Capsular Saccharide of the Invention

The term “saccharide” throughout this specification may indicatepolysaccharide or oligosaccharide and includes both. In frequentembodiments, the saccharide is a polysaccharide, in particular a S.pneumoniae capsular polysaccharide.

Capsular polysaccharides are prepared by standard techniques known tothose of ordinary skill in the art.

In the present invention, capsular polysaccharides may be prepared,e.g., from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14,15B, 18C, 19A, 19F, 22F, 23F and 33F of S. pneumoniae. Typicallycapsular polysaccharides are produced by growing each S. pneumoniaeserotype in a medium (e.g., in a soy-based medium), the polysaccharidesare then prepared from the bacteria culture. Bacterial strains of S.pneumoniae used to make the respective polysaccharides that are used inthe glycoconjugates of the invention may be obtained from establishedculture collections or clinical specimens.

The population of the organism (each S. pneumoniae serotype) is oftenscaled up from a seed vial to seed bottles and passaged through one ormore seed fermentors of increasing volume until production scalefermentation volumes are reached. At the end of the growth cycle thecells are lysed and the lysate broth is then harvested for downstream(purification) processing (see for example WO 2006/110381, WO2008/118752, and U.S. Patent App. Pub. Nos. 2006/0228380, 2006/0228381,2008/0102498 and 2008/0286838).

The individual polysaccharides are typically purified throughcentrifugation, precipitation, ultra-filtration, and/or columnchromatography (see for example WO 2006/110352 and WO 2008/118752).

Purified polysaccharides may be activated (e.g., chemically activated)to make them capable of reacting (e.g., with the eTEC spacer) and thenincorporated into glycoconjugates of the invention, as further describedherein.

S. pneumoniae capsular polysaccharides comprise repeatingoligosaccharide units which may contain up to 8 sugar residues.

In an embodiment, capsular saccharide of the invention may be oneoligosaccharide unit or a shorter than native length saccharide chain ofrepeating oligosaccharide units.

In an embodiment, capsular saccharide of the invention is one repeatingoligosaccharide unit of the relevant serotype.

In an embodiment, capsular saccharide of the invention may beoligosaccharides. Oligosaccharides have a low number of repeat units(typically 5-15 repeat units) and are typically derived synthetically orby hydrolysis of polysaccharides.

Preferably though, all of the capsular saccharides of the presentinvention and in the immunogenic compositions of the present inventionare polysaccharides. High molecular weight capsular polysaccharides areable to induce certain antibody immune responses due to the epitopespresent on the antigenic surface. The isolation and purification of highmolecular weight capsular polysaccharides is preferably contemplated foruse in the conjugates, compositions and methods of the presentinvention.

In some embodiments, the purified polysaccharides before conjugationhave a molecular weight of between 10 kDa and 4,000 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 50 kDaand 4,000 kDa. In further such embodiments, the polysaccharide has amolecular weight of between 50 kDa and 3,500 kDa; between 50 kDa and3,000 kDa; between 50 kDa and 2,500 kDa; between 50 kDa and 2,000 kDa;between 50 kDa and 1,750 kDa; between 50 kDa and 1,500 kDa; between 50kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50 kDa and 750kDa; between 50 kDa and 500 kDa; between 100 kDa and 4,000 kDa; between100 kDa and 3,500 kDa; 100 kDa and 3,000 kDa; 100 kDa and 2,500 kDa; 100kDa and 2,000 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 4,000 kDa; between 200 kDa and3,500 kDa; between 200 kDa and 3,000 kDa; between 200 kDa and 2,500 kDa;between 200 kDa and 2,000 kDa; between 200 kDa and 2,000 kDa; between200 kDa and 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDaand 1,250 kDa; between 200 kDa and 1,000 kDa; between 200 kDa and 750kDa; or between 200 kDa and 500 kDa. Any whole number integer within anyof the above ranges is contemplated as an embodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.Mechanical or chemical sizing maybe employed. Chemical hydrolysis maybeconducted using acetic acid. Mechanical sizing maybe conducted usingHigh Pressure Homogenization Shearing. The molecular weight rangesmentioned above refer to purified polysaccharides before conjugation(e.g., before activation).

In a preferred embodiment the purified polysaccharides, are capsularpolysaccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,12F, 14, 15B, 18C, 19A, 19F, 22F, 23F or 33F of S. pneumoniae, whereinthe capsular polysaccharide has a molecular weight falling within one ofthe molecular weight ranges as described here above.

As used herein, the term “molecular weight” of polysaccharide or ofcarrier protein-polysaccharide conjugate refers to molecular weightcalculated by size exclusion chromatography (SEC) combined withmultiangle laser light scattering detector (MALLS).

In some embodiments, the pneumococcal saccharides from serotypes 9V,18C, 11A, 15B, 22F and/or 33F of the invention are O-acetylated. In someembodiments, the pneumococcal saccharides from serotypes 9V, 11A, 15B,22F and/or 33F of the invention are O-acetylated.

The purified polysaccharides described herein are chemically activatedto make the saccharides capable of reacting with the carrier protein.These pneumococcal conjugates are prepared by separate processes andformulated into a single dosage formulation as described below.

1.2.1 Pneumococcal Polysaccharide from S. pneumoniae Serotypes 1, 3, 4,5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F

Capsular saccharides from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F and 23F may be prepared by standard techniquesknown to those of ordinary skill in the art (see for example WO2006/110381). Capsular polysaccharides can be produced by growing eachS. pneumoniae serotype in a medium; at the end of the growth cycle thecells are lysed and the lysate broth is then harvested for downstream(purification) processing. The individual polysaccharides are typicallypurified through centrifugation, precipitation, ultra-filtration, and/orcolumn chromatography (see for example WO 2006/110352 and WO2008/118752). Purified polysaccharides may be further processed asfurther described herein to prepare glycoconjugates of the invention.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and/or 23Fbefore conjugation have a molecular weight of between 10 kDa and 4,000kDa. In other such embodiments, the polysaccharide has a molecularweight of between 50 kDa and 4,000 kDa; between 50 kDa and 3,000 kDa orbetween 50 kDa and 2,000 kDa. In further such embodiments, thepolysaccharide has a molecular weight of between 50 kDa and 3,500 kDa;between 50 kDa and 3,000 kDa; between 50 kDa and 2,500 kDa; between 50kDa and 2,000 kDa; 50 kDa and 1,750 kDa; between 50 kDa and 1,500 kDa;between 50 kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50kDa and 750 kDa; between 50 kDa and 500 kDa; between 100 kDa and 4,000kDa; between 100 kDa and 3,500 kDa; between 100 kDa and 3,000 kDa;between 100 kDa and 2,500 kDa; between 100 kDa and 2,000 kDa; between100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDaand 1,250 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750kDa; between 100 kDa and 500 kDa; between 200 kDa and 4,000 kDa; between200 kDa and 3,500 kDa; between 200 kDa and 3,000 kDa; between 200 kDaand 2,500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,750kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa. Any whole number integer within any of the aboveranges is contemplated as an embodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

In some embodiments, the pneumococcal saccharides from serotypes 9Vand/or 18C of the invention are O-acetylated. In some embodiments, thepneumococcal saccharide from serotype 9V of the invention isO-acetylated and the pneumococcal saccharide from serotype 18C of theinvention is de-O-acetylated.

1.2.2 Pneumococcal Polysaccharide Serotype 8

The polysaccharide repeating unit of serotype 8 consists of a lineartetrasaccharide unit with one glucuronic acid (GlcpA), twoglucopyranoses (Glcp) and one galactopyranose (Galp) (Jones et al.(1957) The Journal of the American Chemical Society. 79(11):2787-2793).All four monosaccharides are linked via 1,4-linkages as shown at FIG. 1.Serotype 8 saccharides can be obtained directly from bacteria usingisolation procedures known to one of ordinary skill in the art (see forexample methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380,2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498and WO 2008/118752). In addition, they can be produced using syntheticprotocols.

Serotype 8 S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 8 before conjugation have a molecular weight of between 10 kDaand 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 70 kDa and900 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 100 kDa and 800 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa;150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 200 kDa to600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 250 kDa to 600; 250 kDato 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 600 kDa;300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 600 kDa; 500 kDa to600 kDa; and similar desired molecular weight ranges. Any whole numberinteger within any of the above ranges is contemplated as an embodimentof the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

1.2.3 Pneumococcal Polysaccharide Serotype 10A

The polysaccharide repeating unit of serotype 10A consists of a branchedhexasaccharide repeat unit with two galactofuranoses (Gal_(f)), threegalactopyranoses (Gal_(p)), one N-acetylgalactosamine (Gal_(p)NAc) and abackbone phosphoribitol (Jones, C. (1995) Carbohydrate Research269(1):175-181). There are two branching monosaccharides at theβ-GalpNAc moiety (a β-3-Galp and a β-6-Galt) as shown at FIG. 2.

Serotype 10A saccharides can be obtained directly from bacteria usingisolation procedures known to one of ordinary skill in the art (see forexample methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380,2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498and WO 2008/118752). In addition, they can be produced using syntheticprotocols.

Serotype 10A S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 10A before conjugation have a molecular weight of between 10kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 70 kDa and900 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 100 kDa and 800 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa;150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 200 kDa to600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 250 kDa to 600 kDa; 250kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 600kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 600 kDa; 500 kDato 600 kDa; and similar desired molecular weight ranges. Any wholenumber integer within any of the above ranges is contemplated as anembodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

1.2.4 Pneumococcal Polysaccharide Serotype 11A

The polysaccharide repeating unit of serotype 11A consists of a lineartetrasaccharide backbone (two galactopyranoses (Gal_(p)) and twoglucopyranose (Glc_(p)) and a pendent phosphoglycerol (Richards et al.(1988) Adv. Exp. Med. Biol. 228:595-597), as shown at FIG. 3. Thepolysaccharide is O-acetylated at multiple locations and, based on thereported data in the literature (Calix et al. (2011) J Bacteriol.193(19):5271-5278), the total amount of O-acetylation in 11Apolysaccharide is about 2.6 O-acetyl groups per polysaccharide repeatunit.

Serotype 11A saccharides can be obtained directly from bacteria usingisolation procedures known to one of ordinary skill in the art (see forexample methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380,2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498and WO 2008/118752). In addition, they can be produced using syntheticprotocols.

Serotype 11A S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

The isolated serotype 11A capsular polysaccharide obtained bypurification of serotype 11A polysaccharide from the S. pneumoniaelysate and optionally sizing of the purified polysaccharide may becharacterized by different attributes including, for example, themolecular weight (MW) and the mM of acetate per mM of said serotype 11Acapsular polysaccharide.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 11A before conjugation have a molecular weight of between 10kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 70 kDa and900 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 100 kDa and 800 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa;100 kDa to 300 kDa; 100 kDa to 200 kDa; 150 kDa to 600 kDa; 150 kDa to500 kDa; 150 kDa to 400 kDa; 150 kDa to 300 kDa; 150 kDa to 200 kDa; 200kDa to 600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 250 kDa to 600kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDato 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 600 kDa;500 kDa to 600 kDa; and similar desired molecular weight ranges. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

In an embodiment, the size of the purified serotype 11A polysaccharideis reduced by high pressure homogenization. High pressure homogenizationachieves high shear rates by pumping the process stream through a flowpath with sufficiently small dimensions. The shear rate is increased byusing a larger applied homogenization pressure, and exposure time can beincreased by recirculating the feed stream through the homogenizer.

The high pressure homogenization process is particularly appropriate forreducing the size of the purified serotype 11A polysaccharide whilepreserving the structural features of the polysaccharide, such as thepresence of O-acetyl groups.

The presence of O-acetyl in a purified, isolated or activated serotype11A capsular polysaccharide or in a serotype 11A polysaccharide-carrierprotein conjugate is expressed as the number of mM of acetate per mM ofsaid polysaccharide or as the number of O-acetyl group perpolysaccharide repeating unit.

In a preferred embodiment, the purified polysaccharides from S.pneumoniae serotype 11A has at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4or 1.6, μmol acetate per μmol of said serotype 11A capsularpolysaccharide.

1.2.5 Pneumococcal Polysaccharide Serotype 12F

The polysaccharide repeating unit of serotype 12F consists of a lineartrisaccharide backbone (one N-acetylfucosamine (Fuc_(p)NAc), oneN-acetylgalactosamine (Gal_(p)NAc) and one N-acetylmannuronic acid(Man_(p)NAcA)) with two branches: a pendant α-galactopyranose (Gal_(p))linked at C3 of Fuc_(p)NAc and an α-Glc_(p)-(1→2)-α-Glc_(p) disaccharidebranch linked at C3 of Man_(p)NAcA (Leontein et al. (1983) CarbohydrateResearch 114(2):257-266) as shown at FIG. 4.

Serotype 12F Streptococcus pneumoniae strains may be obtained fromestablished culture collections (such as for example the StreptococcalReference Laboratory (Centers for Disease Control and Prevention,Atlanta, Ga.)) or clinical specimens.

Capsular saccharides from S. pneumoniae serotype 12F are prepared bystandard techniques known to those of ordinary skill in the art.Typically capsular polysaccharides are produced by growing each S.pneumoniae serotype in a medium (e.g., in a soy-based medium), thepolysaccharides are then prepared from the bacteria culture. Thepopulation of the organism (S. pneumoniae serotype 12F) is often scaledup from a seed vial to seed bottles and passaged through one or moreseed fermentors of increasing volume until production scale fermentationvolumes are reached. At the end of the growth cycle the cells are lysedand the lysate broth is then harvested for downstream (purification)processing (see for example WO 2006/110381 and WO 2008/118752, U.S.Patent App. Pub. Nos. 2006/0228380, 2006/0228381, 2008/0102498 andUS2008/0286838). The polysaccharides are typically purified throughcentrifugation, precipitation, ultra-filtration, and/or columnchromatography (see for example WO 2006/110352 and WO 2008/118752).

Purified polysaccharides from serotype 12F may be activated (e.g.,chemically activated) to make them capable of reacting and thenincorporated into glycoconjugates of the invention, as further describedherein.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 12F before conjugation have a molecular weight of between 10kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 50 kDa and300 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 70 kDa and 300 kDa. In further embodiments,the capsular polysaccharide has a molecular weight of 90 kDa to 250 kDa;90 kDa to 150 kDa; 90 kDa to 120 kDa; 80 kDa to 120 kDa; 70 kDa to 100kDa; 70 kDa to 110 kDa; 70 kDa to 120 kDa; 70 kDa to 130 kDa; 70 kDa to140 kDa; 70 kDa to 150 kDa; 70 kDa to 160 kDa; 80 kDa to 110 kDa; 80 kDato 120 kDa; 80 kDa to 130 kDa; 80 kDa to 140 kDa; 80 kDa to 150 kDa; 80kDa to 160 kDa; 90 kDa to 110 kDa; 90 kDa to 120 kDa; 90 kDa to 130 kDa;90 kDa to 140 kDa; 90 kDa to 150 kDa; 90 kDa to 160 kDa; 100 kDa to 120kDa; 100 kDa to 130 kDa; 100 kDa to 140 kDa; 100 kDa to 150 kDa; 100 kDato 160 kDa; and similar desired molecular weight ranges. Any wholenumber integer within any of the above ranges is contemplated as anembodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

1.2.6 Pneumococcal Polysaccharide Serotype 15B

As shown at FIG. 5, the polysaccharide repeating unit of serotype 15Bconsists of a branched trisaccharide backbone (one N-acetylglucosamine(Glc_(p)NAc), one galactopyranose (Gal_(p)) and one glucopyranose(Glc_(p))) with an αGal_(p)-βGal_(p) disaccharide branch linked to theC4 hydroxyl group of Glc_(p)NAc. The phosphoglycerol is linked to the C3hydroxyl group of the βGal_(p) residue in the disaccharide branch (Joneset al. (2005) Carbohydrate Research 340(3):403-409). Capsularpolysaccharide from serotype 15C serotype has the identical backbonestructure as serotype 15B but lacks the O-acetylation.

Serotype 15B polysaccharides can be obtained directly from bacteriausing isolation procedures known to one of ordinary skill in the art(see for example methods disclosed in U.S. Patent App. Pub. Nos.2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340,and 2008/0102498 and WO 2008/118752). They can also be produced usingsynthetic protocols known to the man skilled in the art.

Serotype 15B S. pneumoniae strains may be obtained from establishedculture collections (such as for example the American Type CultureCollection (ATCC, Manassas, Va. USA) (e.g., deposit strain No.ATCC10354) or the Streptococcal Reference Laboratory (Centers forDisease Control and Prevention, Atlanta, Ga. USA)) or from clinicalspecimens.

The bacterial cells are grown in a medium, preferably in a soy basedmedium. Following fermentation of bacterial cells that produce S.pneumoniae serotype 15B capsular polysaccharides, the bacterial cellsare lysed to produce a cell lysate. The serotype 15B polysaccharide maythen be isolated from the cell lysate using purification techniquesknown in the art, including the use of centrifugation, depth filtration,precipitation, ultra-filtration, treatment with activate carbon,diafiltration and/or column chromatography (see, for example, U.S.Patent App. Pub. Nos. 2006/0228380, 2006/0228381, 2007/0184071,2007/0184072, 2007/0231340, and 2008/0102498 and WO 2008/118752). Thepurified serotype 15B capsular polysaccharide can then be used for thepreparation of immunogenic conjugates.

The isolated serotype 15B capsular polysaccharide obtained bypurification of serotype 15B polysaccharide from the S. pneumoniaelysate and optionally sizing of the purified polysaccharide can becharacterized by different parameters including, for example, themolecular weight (MW), the mM of acetate per mM of said serotype 15Bcapsular polysaccharide and the mM of glycerol per mM of said serotype15B capsular polysaccharide.

Preferably, in order to generate 15B conjugates with advantageousfilterability characteristics and/or yields, sizing of thepolysaccharide to a target molecular weight range is performed prior tothe conjugation to a carrier protein. Advantageously, the size of thepurified serotype 15B polysaccharide is reduced while preservingcritical features of the structure of the polysaccharide such as forexample the presence of O-acetyl groups. Preferably, the size of thepurified serotype 15B polysaccharide is reduced by mechanicalhomogenization.

In a preferred embodiment, the size of the purified serotype 15Bpolysaccharide is reduced by high pressure homogenization. High pressurehomogenization achieves high shear rates by pumping the process streamthrough a flow path with sufficiently small dimensions. The shear rateis increased by using a larger applied homogenization pressure, andexposure time can be increased by recirculating the feed stream throughthe homogenizer.

The high pressure homogenization process is particularly appropriate forreducing the size of the purified serotype 15B polysaccharide whilepreserving the structural features of the polysaccharide, such as thepresence of O-acetyl groups.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 5 kDa and 500 kDa, between50 kDa and 500 kDa, between 50 kDa and 450 kDa, between 100 kDa and 400kDa, and between 100 kDa and 350 kDa. In a preferred embodiment, theisolated serotype 15B capsular polysaccharide has a molecular weightbetween 100 kDa and 350 kDa. In a preferred embodiment, the isolatedserotype 15B capsular polysaccharide has a molecular weight between 100kDa and 300 kDa. In a preferred embodiment, the isolated serotype 15Bcapsular polysaccharide has a molecular weight between 150 kDa and 300kDa. In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 350 kDa. Infurther embodiments, the capsular polysaccharide has a molecular weightof 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 100 kDato 200 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 150 kDa to 300 kDa;150 kDa to 200 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 250 kDa to500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 500 kDa; 300kDa to 400 kDa; and similar desired molecular weight ranges. Any wholenumber integer within any of the above ranges is contemplated as anembodiment of the disclosure.

Serotype 15B polysaccharide is O-acetylated and the total amount ofO-acetylation is approximately 0.8-0.9 O-acetyl groups perpolysaccharide repeating unit. The degree of O-acetylation of thepolysaccharide can be determined by any method known in the art, forexample, by proton NMR (see for example Lemercinier et al. (1996)Carbohydrate Research 296:83-96; Jones et al. (2002) J. Pharmaceuticaland Biomedical Analysis 30:1233-1247; WO 2005/033148 and WO 00/56357).Another commonly used method is described in Hestrin, S. (1949) J. Biol.Chem. 180:249-261. Preferably, the presence of O-acetyl groups isdetermined by ion-HPLC analysis.

The presence of O-acetyl in a purified, isolated or activated serotype15B capsular polysaccharide or in a serotype 15B polysaccharide-carrierprotein conjugate is expressed as the number of mM of acetate per mM ofsaid polysaccharide or as the number of O-acetyl group perpolysaccharide repeating unit.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or0.8 mM acetate per mM of said serotype 15B capsular polysaccharide. In apreferred embodiment, the isolated serotype 15B capsular polysaccharidecomprises at least 0.5, 0.6 or 0.7 mM acetate per mM of said serotype15B capsular polysaccharide. In a preferred embodiment, the isolatedserotype 15B capsular polysaccharide comprises at least 0.6 mM acetateper mM of said serotype 15B capsular polysaccharide. In a preferredembodiment, the isolated serotype 15B capsular polysaccharide comprisesat least 0.7 mM acetate per mM of said serotype 15B capsularpolysaccharide.

The presence of glycerolphosphate side chains is determined bymeasurement of glycerol using high performance anion exchangechromatography with pulsed amperometric detection (HPAEC-PAD) after itsrelease by treatment of the polysaccharide with hydrofluoric acid (HF).The presence of glycerol in a purified, isolated or activated serotype15B polysaccharide or in a serotype 15B polysaccharide-carrier proteinconjugate is expressed as the number of mM of glycerol per mM ofserotype 15B polysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or0.8 mM glycerol per mM of said serotype 15B capsular polysaccharide. Ina preferred embodiment, the isolated serotype 15B capsularpolysaccharide comprises at least 0.5, 0.6 or 0.7 mM glycerol per mM ofsaid serotype 15B capsular polysaccharide. In a preferred embodiment,the isolated serotype 15B capsular polysaccharide comprises at least 0.6mM glycerol per mM of said serotype 15B capsular polysaccharide. In apreferred embodiment, the isolated serotype 15B capsular polysaccharidecomprises at least 0.7 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 350 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 350 kDa andcomprises at least 0.6 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 300 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 300 kDa andcomprises at least 0.6 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 350 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 350 kDa andcomprises at least 0.6 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide comprises at least 0.6 mM acetate per mM of said serotype15B capsular polysaccharide and at least 0.6 mM glycerol per mM of saidserotype 15B capsular polysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 350 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide and at least 0.6 mM glycerol per mM of said serotype 15Bcapsular polysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 300 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide and at least 0.6 mM glycerol per mM of said serotype 15Bcapsular polysaccharide.

In a preferred embodiment, the isolated serotype 15B capsularpolysaccharide has a molecular weight between 150 kDa and 350 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide and at least 0.6 mM glycerol per mM of said serotype 15Bcapsular polysaccharide.

1.2.7 Pneumococcal Polysaccharide Serotype 22F

As shown at FIG. 6, the polysaccharide repeating unit of serotype 22Fconsists of a branched pentasaccharide backbone (one glucuronic acid(GlcA), one glucopyranose (Glc_(p)), one galactofuranose (Gal_(f)) andtwo rhamnopyranoses (Rha_(p))) with a αGlc_(p) branch linked to the C3hydroxyl group of βRha_(p) (Richards et al. (1989) Canadian Journal ofChemistry 67(6):1038-1050). Approximately 80% of the C2 hydroxyl groupsof the βRha_(p) residue in the polysaccharide repeating unit areO-acetylated. Serotype 22F polysaccharides can be obtained directly frombacteria using isolation procedures known to one of ordinary skill inthe art (see for example methods disclosed in U.S. Patent App. Pub. Nos.2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340,and 2008/0102498 and WO 2008/118752). In addition, they can be producedusing synthetic protocols.

Serotype 22F S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

The isolated serotype 22F capsular polysaccharide obtained bypurification of serotype 22F polysaccharide from the S. pneumoniaelysate and optionally sizing of the purified polysaccharide can becharacterized by different parameters including, for example, themolecular weight (MW) and the mM of acetate per mM of said serotype 22Fcapsular polysaccharide.

Preferably, in order to generate serotype 22F conjugates withadvantageous filterability characteristics and/or yields, sizing of thepolysaccharide to a target molecular weight range is performed prior tothe conjugation to a carrier protein. Advantageously, the size of thepurified serotype 22F polysaccharide is reduced while preservingcritical features of the structure of the polysaccharide such as forexample the presence of O-acetyl group. Preferably, the size of thepurified serotype 22F polysaccharide is reduced by mechanicalhomogenization.

In a preferred embodiment, the size of the purified polysaccharide isreduced by high pressure homogenization. High pressure homogenizationachieves high shear rates by pumping the process stream through a flowpath with sufficiently small dimensions. The shear rate is increased byusing a larger applied homogenization pressure, and exposure time can beincreased by recirculating the feed stream through the homogenizer.

The high pressure homogenization process is particularly appropriate forreducing the size of the purified serotype 22F polysaccharide whilepreserving the structural features of the polysaccharide, such as thepresence of O-acetyl groups.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 22F before conjugation have a molecular weight of between 10kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 70 kDa to900 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 100 kDa to 800 kDa. In another embodiment,the capsular polysaccharide has a molecular weight of between 200 kDa to600 kDa. In another embodiment, the capsular polysaccharide has amolecular weight of between 400 kDa to 700 kDa.

In further embodiments, the capsular polysaccharide has a molecularweight of 100 kDa to 1,000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa;100 kDa to 700 kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to400 kDa; 100 kDa to 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa;150 kDa to 800 kDa; 150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to500 kDa; 150 kDa to 400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa;200 kDa to 900 kDa; 200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 200 kDa to 300 kDa; 250kDa to 1,000 kDa; 250 kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700kDa; 250 kDa to 600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDato 350 kDa; 300 kDa to 1,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800kDa; 300 kDa to 700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDato 400 kDa; 400 kDa to 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800kDa; 400 kDa to 700 kDa; 400 kDa to 600 kDa; 500 kDa to 600 kDa; andsimilar desired molecular weight ranges. Any whole number integer withinany of the above ranges is contemplated as an embodiment of thedisclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described hereabove, 22Fpolysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

The degree of O-acetylation of the polysaccharide can be determined byany method known in the art, for example, by proton NMR (Lemercinier etal. (1996) Carbohydrate Research 296:83-96; Jones et al. (2002) J.Pharmaceutical and Biomedical Analysis 30:1233-1247; WO 2005/033148 andWO 00/56357). Another commonly used method is described in Hestrin, S.(1949) J. Biol. Chem. 180:249-261. Preferably, the presence of O-acetylgroups is determined by ion-HPLC analysis.

The presence of O-acetyl in a purified, isolated or activated serotype22F capsular polysaccharide or in a serotype 22F polysaccharide-carrierprotein conjugate is expressed as the number of mM of acetate per mM ofsaid polysaccharide or as the number of O-acetyl group perpolysaccharide repeating unit.

In a preferred embodiment, the purified polysaccharides from S.pneumoniae serotype 22F has at least 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4 or1.6, μmol acetate per μmol of said serotype 22F capsular polysaccharide.

1.2.8 Pneumococcal Polysaccharide Serotype 33F

As shown at FIG. 7, the polysaccharide repeating unit of serotype 33Fconsists of a branched pentasaccharide backbone (two galactopyranoses(Gal_(p)), two galactofuranoses (Gal_(f)) and one glucopyranose(Glc_(p)) with a terminal αGal_(p) linked to the C2 hydroxyl group ofαGal_(p) residue within the backbone (Lemercinier et al. (2006)Carbohydrate Research 341(1):68-74). It has been reported in theliterature that the C2 hydroxyl group of the backbone 3-β-Gal_(f)residue is O-acetylated.

Serotype 33F polysaccharides can be obtained directly from bacteriausing isolation procedures known to one of ordinary skill in the art(see for example methods disclosed in U.S. Patent App. Pub. Nos.2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340,and 2008/0102498 and WO 2008/118752). In addition, they can be producedusing synthetic protocols.

Serotype 33F S. pneumoniae strains may be obtained from establishedculture collections (such as for example the Streptococcal ReferenceLaboratory (Centers for Disease Control and Prevention, Atlanta, Ga.))or clinical specimens.

Purified polysaccharides from serotype 33F may be activated (e.g.,chemically activated) to make them capable of reacting and thenincorporated into glycoconjugates of the invention, as further describedherein.

The isolated serotype 33F capsular polysaccharide obtained bypurification of serotype 33F polysaccharide from the S. pneumoniaelysate and optionally sizing of the purified polysaccharide can becharacterized by different parameters including, for example, themolecular weight and the mM of acetate per mM of said serotype 33Fcapsular polysaccharide.

In some embodiments, the purified polysaccharides from S. pneumoniaeserotype 33F before conjugation have a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 2,000 kDa. In further suchembodiments, the saccharide has a molecular weight of between 50 kDa and1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa;between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDaand 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa. Any whole number integer within any of the aboveranges is contemplated as an embodiment of the disclosure.

A polysaccharide can become slightly reduced in size during normalpurification procedures. Additionally, as described herein,polysaccharide can be subjected to sizing techniques before conjugation.The molecular weight ranges mentioned above refer to purifiedpolysaccharides before conjugation (e.g., before activation) after aneventual sizing step.

The presence of O-acetyl in a purified, isolated or activated serotype33F capsular polysaccharide or in a serotype 33F polysaccharide-carrierprotein conjugate is expressed as the number of mM of acetate per mM ofsaid polysaccharide or as the number of O-acetyl group perpolysaccharide repeating unit.

In a preferred embodiment, the purified polysaccharides from S.pneumoniae serotype 33F has at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4or 1.6, μmol acetate per μmol of said serotype 33F capsularpolysaccharide.

1.3 Glycoconjugates of the Invention

The purified saccharides are chemically activated to make thesaccharides (i.e., activated saccharides) capable of reacting with thecarrier protein. Once activated, each capsular saccharide is separatelyconjugated to a carrier protein to form a glycoconjugate. In oneembodiment, each capsular saccharide is conjugated to the same carrierprotein. The chemical activation of the saccharides and subsequentconjugation to the carrier protein can be achieved by the activation andconjugation methods disclosed herein.

1.3.1 Glycoconjugates from S. pneumoniae Serotype 1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F and 23F

Capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F of S. pneumoniae are prepared by standardtechniques known to those of ordinary skill in the art (see for exampleWO 2006/110381, WO 2008/118752, WO 2006/110352, and U.S. Patent App.Pub. Nos. 2006/0228380, 2006/0228381, 2008/0102498 and 2008/0286838).

In an embodiment, the polysaccharides are activated with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated polysaccharide is then coupled directly orvia a spacer (linker) group to an amino group on the carrier protein(preferably CRM₁₉₇). For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example usingN-[γ-maleimidobutyrloxy]succinimide ester (GMBS)) or a haloacetylatedcarrier protein (for example using iodoacetimide, N-succinimidylbromoacetate (SBA; SIB), N-succinimidyl(4-iodoacetyl)aminobenzoate(SIAB), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB),N-succinimidyl iodoacetate (SIA) or succinimidyl3-[bromoacetamido]proprionate (SBAP)). Preferably, the cyanate ester(optionally made by CDAP chemistry) is coupled with hexane diamine oradipic acid dihydrazide (ADH) and the amino-derivatised saccharide isconjugated to the carrier protein (e.g., CRM₁₉₇) using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/29094.

Other suitable techniques for conjugation use carbodiimides, hydrazides,active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,S-NHS, EDC, TSTU. Many are described in International Patent ApplicationPublication No. WO 98/42721. Conjugation may involve a carbonyl linkerwhich may be formed by reaction of a free hydroxyl group of thesaccharide with 1,1′-carbonyldiimidazole (CDI) (see Bethell et al.(1979) J. Biol. Chem. 254:2572-2574; Hearn et al. (1981) J. Chromatogr.218:509-518) followed by reaction with a protein to form a carbamatelinkage. This may involve reduction of the anomeric terminus to aprimary hydroxyl group, optional protection/deprotection of the primaryhydroxyl group, reaction of the primary hydroxyl group with CDI to forma CDI carbamate intermediate and coupling the CDI carbamate intermediatewith an amino group on a protein.

In an preferred embodiment, at least one of capsular polysaccharidesfrom serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F ofS. pneumoniae is conjugated to the carrier protein by reductiveamination (such as described in U.S. Patent Appl. Pub. Nos.2006/0228380, 2007/0231340, 2007/0184071 and 2007/0184072, WO2006/110381, WO 2008/079653, and WO 2008/143709). In a preferredembodiment, the capsular polysaccharides from serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F and 23F of S. pneumoniae are allconjugated to the carrier protein by reductive amination.

Reductive amination involves two steps, (1) oxidation of thepolysaccharide, (2) reduction of the activated polysaccharide and acarrier protein to form a conjugate. Before oxidation, thepolysaccharide is optionally hydrolyzed. Mechanical or chemicalhydrolysis may be employed. Chemical hydrolysis may be conducted usingacetic acid. The oxidation step may involve reaction with periodate. Forthe purpose of the present invention, the term “periodate” includes bothperiodate and periodic acid; the term also includes both metaperiodate(IO₄ ⁻) and orthoperiodate (IO₆ ⁵⁻) and the various salts of periodate(e.g., sodium periodate and potassium periodate).

In an embodiment the capsular polysaccharide from serotype 1, 3, 4, 5,6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F of S. pneumoniae is oxidized inthe presence of metaperiodate, preferably in the presence of sodiumperiodate (NaIO₄). In another embodiment the capsular polysaccharidesfrom serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F ofS. pneumoniae is oxydized in the presence of orthoperiodate, preferablyin the presence of periodic acid.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to as “activated polysaccharide”here below. The activated polysaccharide and the carrier protein may belyophilised (freeze-dried), either independently (discretelyophilization) or together (co-lyophilized). In one embodiment theactivated polysaccharide and the carrier protein are co-lyophilized. Inanother embodiment the activated polysaccharide and the carrier proteinare lyophilized independently.

In one embodiment the lyophilization takes place in the presence of anon-reducing sugar, possible non-reducing sugars include sucrose,trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitoland palatinit.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(so-called reductive amination), using a reducing agent. Reducing agentswhich are suitable include the cyanoborohydrides, such as sodiumcyanoborohydride, borane-pyridine, or borohydride exchange resin. In oneembodiment the reducing agent is sodium cyanoborohydride. In anembodiment, the reduction reaction is carried out in aqueous solvent, inanother embodiment the reaction is carried out in aprotic solvent. In anembodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilized.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄). Following the conjugation (the reduction reactionand optionally the capping), the glycoconjugates may be purified. Theglycoconjugates maybe purified by diafiltration and/or ion exchangechromatography and/or size exclusion chromatography. In an embodiment,the glycoconjugates are purified by diafiltration or ion exchangechromatography or size exclusion chromatography. In one embodiment theglycoconjugates are sterile filtered. In some embodiments, theglycoconjugate from S. pneumoniae serotypes 9V and/or 18C comprise asaccharide which has a degree of O-acetylation of between 10% and 100%,between 20% and 100%, between 30% and 100%, between 40% and 100%,between 50% and 100%, between 60% and 100%, between 70% and 100%,between 75% and 100%, between 80% and 100%, between 90% and 100%,between 50% and 90%, between 60% and 90%, between 70% and 90% or between80% and 90%. In other embodiments, the degree of O-acetylation is ≥10%,≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, or ≥90%, or about 100%.

In some embodiments, the glycoconjugate from S. pneumoniae serotypes 9Vand/or 18C of the invention are O-acetylated. In some embodiments, theglycoconjugate from S. pneumoniae serotype 9V is O-acetylated and theglycoconjugate from S. pneumoniae serotype 18C is de-O-acetylated.

1.3.2 Glycoconjugates from S. pneumoniae Serotype 22F

In an embodiment, the serotype 22F glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB,SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/29094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 22F glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps, (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit, (2) reduction of the activated polysaccharide and a carrierprotein (e.g., CRM₁₉₇) to form a conjugate.

Preferably, before oxidation, sizing of the serotype 22F polysaccharideto a target molecular weight (MW) range is performed. Advantageously,the size of the purified serotype 22F polysaccharide is reduced whilepreserving critical features of the structure of the polysaccharide suchas for example the presence of O-acetyl groups. Preferably, the size ofthe purified serotype 22F polysaccharide is reduced by mechanicalhomogenization (see section 1.2.7 above).

In an embodiment, serotype polysaccharide is activated (oxidized) by aprocess comprising the step of:

(a) reacting isolated serotype 22F polysaccharide with an oxidizingagent;

(b) quenching the oxidation reaction by addition of a quenching agentresulting in an activated serotype 22F polysaccharide.

In a preferred embodiment, the oxidizing agent is periodate. For thepurpose of the present invention, the term “periodate” includes bothperiodate and periodic acid; the term also includes both metaperiodate(IO₄ ⁻) and orthoperiodate (IO₆ ⁵⁻) and the various salts of periodate(e.g., sodium periodate and potassium periodate). In a preferredembodiment, the oxidizing agent is sodium periodate. In a preferredembodiment, the periodate used for the oxidation of serotype 22Fpolysaccharide is metaperiodate. In a preferred embodiment the periodateused for the oxidation of serotype 22F polysaccharide is sodiummetaperiodate.

In one embodiment, the quenching agent is selected from vicinal diols,1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid.

In one embodiment, the quenching agent is a 1,2-aminoalcohols of formula(I):

wherein R¹ is selected from H, methyl, ethyl, propyl or isopropyl.

In one embodiment, the quenching agent is selected from sodium andpotassium salts of sulfite, bisulfate, dithionite, metabisulfite,thiosulfate, phosphites, hypophosphites or phosphorous acid.

In one embodiment, the quenching agent is an amino acid. In suchembodiments, said amino acid may be selected from serine, threonine,cysteine, cystine, methionine, proline, hydroxyproline, tryptophan,tyrosine, and histidine.

In one embodiment, the quenching agent is a sulfite such as bisulfate,dithionite, metabisulfite, thiosulfate.

In one embodiment, the quenching agent is a compound comprising twovicinal hydroxyl groups (vicinal diols), i.e., two hydroxyl groupscovalently linked to two adjacent carbon atoms.

Preferably, the quenching agent is a compound of formula (II):

wherein R¹ and R² are each independently selected from H, methyl, ethyl,propyl or isopropyl.

In a preferred embodiment, the quenching agent is glycerol, ethyleneglycol, propan-1,2-diol, butan-1,2-diol or butan-2,3-diol, or ascorbicacid. In a preferred embodiment, the quenching agent is butan-2,3-diol.

In a preferred embodiment, the isolated serotype 22F polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 22F polysaccharide with periodate;

(b) quenching the oxidation reaction by addition of butan-2,3-diolresulting in an activated serotype 22F polysaccharide.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to as “activated polysaccharide”here below.

In a preferred embodiment, the activated serotype 22F polysaccharide ispurified. The activated serotype 22F polysaccharide is purifiedaccording to methods known to the man skilled in the art such as gelpermeation chromatography (GPC), dialysis orultrafiltration/diafiltration. For example, the activated 22Fpolysaccharide is purified by concentration and diafiltration using anultrafiltration device.

In a preferred embodiment the degree of oxidation of the activatedserotype 22F polysaccharide is between 2 and 30, between 2 and 25,between 2 and 20, between 2 and 15, between 2 and 10, between 2 and 5,between 5 and 30, between 5 and 25, between 5 and 20, between 5 and 15,between 5 and 10, between 10 and 30, between 10 and 25, between 10 and20, between 10 and 15, between 15 and 30, between 15 and 25, between 15and 20, between 20 to 30, or between 20 to 25. In a preferred embodimentthe degree of oxidation of the activated serotype 22F polysaccharide isbetween 2 and 10, between 4 and 8, between 4 and 6, between 6 and 8,between 6 and 12, between 8 and 14, between 9 and 11, between 10 and 16,between 12 and 16, between 14 and 18, between 16 and 20, between 16 and18, between 18 and 22, or between 18 and 20.

In a preferred embodiment, the activated serotype 22F polysaccharide hasa molecular weight between 25 kDa and 1,000 kDa, between 100 kDa and1,000 kDa, between 300 kDa and 800 kDa, between 300 kDa and 700 kDa,between 300 kDa and 600 kDa, between 400 kDa and 1,000 kDa, between 400kDa and 800 kDa, between 400 kDa and 700 kDa or between 400 kDa and 600kDa. In an embodiment, the activated serotype 22F polysaccharide has amolecular weight between 300 kDa and 800 kDa. In an embodiment, theactivated serotype 22F polysaccharide has a molecular weight between 400kDa and 600 kDa. In a preferred embodiment, the activated serotype 22Fpolysaccharide has a molecular weight between 400 kDa and 600 kDa and adegree of oxidation between 10 and 25, between 10 and 20, between 12 and20 or between 14 and 18. In a preferred embodiment, the activatedserotype 22F polysaccharide has a molecular weight between 400 kDa and600 kDa and a degree of oxidation between 10 and 20.

In a preferred embodiment, the activated serotype 22F polysaccharidecomprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 or about 0.8 mMacetate per mM serotype 22F polysaccharide. In a preferred embodiment,the activated serotype 22F polysaccharide comprises at least 0.5, 0.6 or0.7 mM acetate per mM serotype 22F polysaccharide. In a preferredembodiment, the activated serotype 22F polysaccharide comprises at least0.6 mM acetate per mM serotype 22F polysaccharide. In a preferredembodiment, the activated serotype 22F polysaccharide comprises at least0.7 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the activated serotype 22F polysaccharide hasa molecular weight between 400 kDa and 800 kDa and comprises at least0.6 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the activated serotype 22F polysaccharide hasa molecular weight between 400 kDa and 800 kDa, a degree of oxidationbetween 12 and 20 and comprises at least 0.6 mM acetate per mM serotype22F polysaccharide.

The activated polysaccharide and/or the carrier protein may belyophilised (freeze-dried), either independently (discretelyophilization) or together (co-lyophilized).

In an embodiment, the activated serotype 22F polysaccharide islyophilized, optionally in the presence of saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. In oneembodiment, the lyophilized activated polysaccharide is then compoundedwith a solution comprising the carrier protein.

In another embodiment the activated polysaccharide and the carrierprotein are co-lyophilised. In such embodiments, the activated serotype22F polysaccharide is compounded with the carrier protein andlyophilized optionally in the presence of a saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. Theco-lyophilized polysaccharide and carrier protein can then beresuspended in solution and reacted with a reducing agent.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(reductive amination), using a reducing agent.

The activated serotype 22F polysaccharide can be conjugated to a carrierprotein by a process comprising the step of:

(c) compounding the activated serotype 22F polysaccharide with a carrierprotein; and

(d) reacting the compounded activated serotype 22F polysaccharide andcarrier protein with a reducing agent to form a serotype 22Fpolysaccharide-carrier protein conjugate.

In an embodiment, the reduction reaction is carried out in aqueoussolvent, in another embodiment the reaction is carried out in aproticsolvent. In an embodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide)) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilised.

The conjugation of activated serotype 22F polysaccharide with a proteincarrier by reductive amination in dimethylsulfoxide (DMSO) is suitableto preserve the O-acetyl content of the polysaccharide as compared, forexample, to reductive amination in aqueous phase where the level ofO-acetylation of the polysaccharide may be significantly reduced.Therefore in a preferred embodiment, step (c) and step (d) are carriedout in DMSO.

In an embodiment, the reducing agent is sodium cyanoborohydride, sodiumtriacetoxyborohydride, sodium or zinc borohydride in the presence ofBronsted or Lewis acids, amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMe^(i)PrN-BH₃, benzylamine-BH₃ or 5-ethyl-2-methylpyridine borane(PEMB). In a preferred embodiment, the reducing agent is sodiumcyanoborohydride.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent

is sodium borohydride (NaBH₄).

Following conjugation of serotype 22F polysaccharide to the carrierprotein, the glycoconjugate can be purified (enriched with respect tothe amount of polysaccharide-protein conjugate) by a variety oftechniques known to the skilled person. These techniques includedialysis, concentration/diafiltration operations, tangential flowfiltration precipitation/elution, column chromatography (DEAE orhydrophobic interaction chromatography), and depth filtration.

In some embodiments, the serotype 22F glycoconjugates of the presentinvention comprise a saccharide having a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 1,000 kDa. In other suchembodiments, the saccharide has a molecular weight of between 70 kDa and900 kDa. In other such embodiments, the saccharide has a molecularweight of between 100 kDa and 800 kDa. In other such embodiments, thesaccharide has a molecular weight of between 200 kDa and 600 kDa. Infurther such embodiments, the saccharide has a molecular weight of 100kDa to 1,000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDato 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to 800kDa; 150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDato 400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900kDa; 200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDato 500 kDa; 200 kDa to 400 kDa; 200 kDa to 300 kDa; 250 kDa to 1,000kDa; 250 kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDato 600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa;300 kDa to 1000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400kDa to 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700kDa; 400 kDa to 600 kDa; 500 kDa to 600 kDa. Any whole number integerwithin any of the above ranges is contemplated as an embodiment of thedisclosure. In some such embodiments, the serotype 22F glycoconjugatesare prepared using reductive amination.

In some embodiments, the serotype 22F glycoconjugate of the inventionhas a molecular weight of between 400 kDa and 15,000 kDa; between 500kDa and 10,000 kDa; between 2,000 kDa and 10,000 kDa; between 3,000 kDaand 8,000 kDa; or between 3,000 kDa and 5,000 kDa. In other embodiments,the serotype 22F glycoconjugate has a molecular weight of between 500kDa and 10,000 kDa. In other embodiments, the serotype 22Fglycoconjugate has a molecular weight of between 1,000 kDa and 8,000kDa. In still other embodiments, the serotype 22F glycoconjugate has amolecular weight of between 2,000 kDa and 8,000 kDa or between 3,000 kDaand 7,000 kDa. In further embodiments, the serotype 22F glycoconjugateof the invention has a molecular weight of between 200 kDa and 20,000kDa; between 200 kDa and 15,000 kDa; between 200 kDa and 10,000 kDa;between 200 kDa and 7,500 kDa; between 200 kDa and 5,000 kDa; between200 kDa and 3,000 kDa; between 200 kDa and 1,000 kDa; between 500 kDaand 20,000 kDa; between 500 kDa and 15,000 kDa; between 500 kDa and12,500 kDa; between 500 kDa and 10,000 kDa; between 500 kDa and 7,500kDa; between 500 kDa and 6,000 kDa; between 500 kDa and 5,000 kDa;between 500 kDa and 4,000 kDa; between 500 kDa and 3,000 kDa; between500 kDa and 2,000 kDa; between 500 kDa and 1,500 kDa; between 500 kDaand 1,000 kDa; between 750 kDa and 20,000 kDa; between 750 kDa and15,000 kDa; between 750 kDa and 12,500 kDa; between 750 kDa and 10,000kDa; between 750 kDa and 7,500 kDa; between 750 kDa and 6,000 kDa;between 750 kDa and 5,000 kDa; between 750 kDa and 4,000 kDa; between750 kDa and 3,000 kDa; between 750 kDa and 2,000 kDa; between 750 kDaand 1,500 kDa; between 1,000 kDa and 15,000 kDa; between 1,000 kDa and12,500 kDa; between 1,000 kDa and 10,000 kDa; between 1,000 kDa and7,500 kDa; between 1,000 kDa and 6,000 kDa; between 1,000 kDa and 5,000kDa; between 1,000 kDa and 4,000 kDa; between 1,000 kDa and 2,500 kDa;between 2,000 kDa and 15,000 kDa; between 2,000 kDa and 12,500 kDa;between 2,000 kDa and 10,000 kDa; between 2,000 kDa and 7,500 kDa;between 2,000 kDa and 6,000 kDa; between 2,000 kDa and 5,000 kDa;between 2,000 kDa and 4,000 kDa; or between 2,000 kDa and 3,000 kDa.

In further embodiments, the serotype 22F glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 15,000 kDa; between 3,000 kDa and 10,000 kDa; between3,000 kDa and 7,500 kDa; between 3,000 kDa and 5,000 kDa; between 4,000kDa and 20,000 kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDaand 12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and5,000 kDa.

In further embodiments, the serotype 22F glycoconjugate of the inventionhas a molecular weight of between 5,000 kDa and 20,000 kDa; between5,000 kDa and 15,000 kDa; between 5,000 kDa and 10,000 kDa; between5,000 kDa and 7,500 kDa; between 6,000 kDa and 20,000 kDa; between 6,000kDa and 15,000 kDa; between 6,000 kDa and 12,500 kDa; between 6,000 kDaand 10,000 kDa or between 6,000 kDa and 7,500 kDa.

The molecular weight of the glycoconjugate is measured by SEC-MALLS. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure.

In a preferred embodiment, the serotype 22F glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 orabout 0.8 mM acetate per mM serotype 22F polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.5, 0.6 or 0.7 mMacetate per mM serotype 22F polysaccharide. In a preferred embodiment,the glycoconjugate comprises at least 0.6 mM acetate per mM serotype 22Fpolysaccharide. In a preferred embodiment, the glycoconjugate comprisesat least 0.7 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the ratio of mM acetate per mM serotype 22Fpolysaccharide in the glycoconjugate to mM acetate per mM serotype 22Fpolysaccharide in the isolated polysaccharide is at least 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferred embodiment, the ratioof mM acetate per mM serotype 22F polysaccharide in the glycoconjugateto mM acetate per mM serotype 22F polysaccharide in the isolatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 22F polysaccharide in the glycoconjugate tomM acetate per mM serotype 22F polysaccharide in the isolatedpolysaccharide is at least 0.9.

In a preferred embodiment, the ratio of mM acetate per mM serotype 22Fpolysaccharide in the glycoconjugate to mM acetate per mM serotype 22Fpolysaccharide in the activated polysaccharide is at least 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferred embodiment, the ratioof mM acetate per mM serotype 22F polysaccharide in the glycoconjugateto mM acetate per mM serotype 22F polysaccharide in the activatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 22F polysaccharide in the glycoconjugate tomM acetate per mM serotype 22F polysaccharide in the activatedpolysaccharide is at least 0.9.

Another way to characterize the serotype 22F glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials. In a preferred embodiment, the degreeof conjugation of the serotype 22F glycoconjugate of the invention isbetween 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8,between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15,between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6,between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10,between 8 and 15, between 8 and 12, between 10 and 15 or between 10 and12. In an embodiment, the degree of conjugation of the serotype 22Fglycoconjugate of the invention is about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14 or about 15. In a preferred embodiment, the degree ofconjugation of the serotype 22F glycoconjugate of the invention isbetween 4 and 7. In some such embodiments, the carrier protein isCRM₁₉₇.

The serotype 22F glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the ratio of serotype 22F polysaccharideto carrier protein in the glycoconjugate (w/w) is between 0.5 and 3.0(e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0,about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3,about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, orabout 3.0). In other embodiments, the saccharide to carrier proteinratio (w/w) is between 0.5 and 2.0, between 0.5 and 1.5, between 0.8 and1.2, between 0.5 and 1.0, between 1.0 and 1.5 or between 1.0 and 2.0. Infurther embodiments, the saccharide to carrier protein ratio (w/w) isbetween 0.8 and 1.2. In a preferred embodiment, the ratio of serotype22F capsular polysaccharide to carrier protein in the conjugate isbetween 0.9 and 1.1. In some such embodiments, the carrier protein isCRM₁₉₇.

The serotype 22F glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In a preferred embodiment, the serotype 22F glycoconjugate comprisesless than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of freeserotype 22F polysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22Fglycoconjugate comprises less than about 40% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22Fglycoconjugate comprises less than about 25% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22Fglycoconjugate comprises less than about 20% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22Fglycoconjugate comprises less than about 15% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide.

The serotype 22F glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate. Size Exclusion Chromatography (SEC) isused in gravity fed columns to profile the molecular size distributionof conjugates. Large molecules excluded from the pores in the mediaelute more quickly than small molecules. Fraction collectors are used tocollect the column eluate. The fractions are tested colorimetrically bysaccharide assay. For the determination of K_(d), columns are calibratedto establish the fraction at which molecules are fully excluded (V₀),(K_(d)=0), and the fraction representing the maximum retention (V_(i)),(K_(d)=1). The fraction at which a specified sample attribute is reached(V_(e)), is related to K_(d) by the expression,K_(d)=(V_(e)−V₀)/(V_(i)−V₀).

In a preferred embodiment, at least 30% of the serotype 22Fglycoconjugate has a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40% of the glycoconjugate has a K_(d)below or equal to 0.3 in a CL-4B column. In a preferred embodiment, atleast 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serotype 22Fglycoconjugate has a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 60% of the serotype 22F glycoconjugatehas a K_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, between 50% and 80% of the serotype 22F glycoconjugate has aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, between 65% and 80% of the serotype 22F glycoconjugate has aK_(d) below or equal to 0.3 in a CL-4B column.

1.3.3 Glycoconjugates from S. pneumoniae Serotype 33F

In an embodiment, the serotype 33F glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/29094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In certain embodiments, the serotype 33F glycoconjugates of theinvention are prepared using reductive amination. In such embodiment,the serotype 33F glycoconjugates of the invention maybe prepared usingreductive amination in aqueous phase (RAC/aqueous). Reductive aminationin aqueous phase has been successfully applied to produce pneumococcalconjugate vaccine (see, e.g., WO 2006/110381). Preferably though, whenusing reductive amination, the serotype 33F glycoconjugates are preparedvia reductive amination in DMSO (RAC/DMSO). In view of the challengesassociated with the preservation of O-acetyl functionality usingRAC/aqueous process, reductive amination in DMSO is preferred. RAC/DMSOhas been successfully applied to produce pneumococcal conjugate vaccine(see, e.g., WO 2006/110381).

In preferred embodiments, the serotype 33F glycoconjugates of theinvention are prepared using eTEC conjugation (hereinafter “serotype 33FeTEC linked glycoconjugates”), such as described at Examples 1, 2 and 3and in WO 2014/027302.

Said 33F glycoconjugates comprise a saccharide covalently conjugated toa carrier protein through one or more eTEC spacers, wherein thesaccharide is covalently conjugated to the eTEC spacer through acarbamate linkage, and wherein the carrier protein is covalentlyconjugated to the eTEC spacer through an amide linkage. The eTEC linkedglycoconjugates of the invention may be represented by the generalformula (III):

wherein the atoms that comprise the eTEC spacer are contained in thecentral box. The eTEC spacer includes seven linear atoms (i.e.,—C(O)NH(CH₂)₂SCH₂C(O)—) and provides stable thioether and amide bondsbetween the saccharide and carrier protein. Synthesis of the eTEC linkedglycoconjugate involves reaction of an activated hydroxyl group of thesaccharide with the amino group of a thioalkylamine reagent, e.g.,cystamine or cysteinamine or a salt thereof, forming a carbamate linkageto the saccharide to provide a thiolated saccharide. Generation of oneor more free sulfhydryl groups is accomplished by reaction with areducing agent to provide an activated thiolated saccharide. Reaction ofthe free sulfhydryl groups of the activated thiolated saccharide with anactivated carrier protein having one or more α-haloacetamide groups onamine containing residues generates a thioether bond to form theconjugate, wherein the carrier protein is attached to the eTEC spacerthrough an amide bond.

In serotype 33F glycoconjugates of the invention, the saccharide may bea polysaccharide or an oligosaccharide. The carrier protein may beselected from any suitable carrier as described herein or known to thoseof skill in the art. In frequent embodiments, the saccharide is apolysaccharide. In some such embodiments, the carrier protein is CRM₁₉₇.In some such embodiments, the eTEC linked glycoconjugate comprises a S.pneumoniae serotype 33F capsular polysaccharide.

In particularly preferred embodiments, the eTEC linked glycoconjugatecomprises a Pn-33F capsular polysaccharide, which is covalentlyconjugated to CRM₁₉₇ through an eTEC spacer (serotype 33F eTEC linkedglycoconjugates).

In some embodiments, the serotype 33F glycoconjugates of the presentinvention comprise a saccharide having a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 2,000 kDa. In further suchembodiments, the saccharide has a molecular weight of between 50 kDa and1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa;between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDaand 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa. Any whole number integer within any of the aboveranges is contemplated as an embodiment of the disclosure.

In some embodiments, the serotype 33F glycoconjugate of the inventionhas a molecular weight of between 50 kDa and 20,000 kDa. In otherembodiments, the serotype 33F glycoconjugate has a molecular weight ofbetween 500 kDa and 10,000 kDa. In other embodiments, the serotype 33Fglycoconjugate has a molecular weight of between 200 kDa and 10,000 kDa.In still other embodiments, the serotype 33F glycoconjugate has amolecular weight of between 1,000 kDa and 3,000 kDa.

In further embodiments, the serotype 33F glycoconjugate of the inventionhas a molecular weight of between 200 kDa and 20,000 kDa; between 200kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between 200 kDa and7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDa and 3,000 kDa;between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa; between 500 kDaand 10,000 kDa; between 500 kDa and 7,500 kDa; between 500 kDa and 6,000kDa; between 500 kDa and 5,000 kDa; between 500 kDa and 4,000 kDa;between 500 kDa and 3,000 kDa; between 500 kDa and 2,000 kDa; between500 kDa and 1,500 kDa; between 500 kDa and 1,000 kDa; between 750 kDaand 20,000 kDa; between 750 kDa and 15,000 kDa; between 750 kDa and12,500 kDa; between 750 kDa and 10,000 kDa; between 750 kDa and 7,500kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000 kDa;between 750 kDa and 4,000 kDa; between 750 kDa and 3,000 kDa; between750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between 1,000 kDaand 15,000 kDa; between 1,000 kDa and 12,500 kDa; between 1,000 kDa and10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa;between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 15,000 kDa;between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa;between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa;between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa;between 2,000 kDa and 3,000 kDa; between 3,000 kDa and 20,000 kDa;between 3,000 kDa and 15,000 kDa; between 3,000 kDa and 12,500 kDa;between 3,000 kDa and 10,000 kDa; between 3,000 kDa and 9,000 kDa;between 3,000 kDa and 8,000 kDa; between 3,000 kDa and 7,000 kDa;between 3,000 kDa and 6,000 kDa; between 3,000 kDa and 5,000 kDa orbetween 3,000 kDa and 4,000 kDa. Any whole number integer within any ofthe above ranges is contemplated as an embodiment of the disclosure.

Another way to characterize the serotype 33F glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide, which can becharacterized as a range of conjugated lysines (degree of conjugation).

In a preferred embodiment, the degree of conjugation of the serotype 33Fglycoconjugate of the invention is between 2 and 20, between 4 and 16,between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8,between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15,between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6,between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10,between 8 and 15, between 8 and 12, between 10 and 15 or between 10 and12. In an embodiment, the degree of conjugation of the serotype 33Fglycoconjugate of the invention is about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19 or about20. In a preferred embodiment, the degree of conjugation of the serotype33F glycoconjugate of the invention is between 4 and 16. In some suchembodiments, the carrier protein is CRM₁₉₇.

In a preferred embodiment, the carrier protein comprises CRM₁₉₇, whichcontains 39 lysine residues. In some such embodiments, the CRM₁₉₇ maycomprise 4 to 16 lysine residues out of 39 covalently linked to thesaccharide. Another way to express this parameter is that about 10% toabout 41% of CRM₁₉₇ lysines are covalently linked to the saccharide. Inanother such embodiment, the CRM₁₉₇ may comprise 2 to 20 lysine residuesout of 39 covalently linked to the saccharide. Another way to expressthis parameter is that about 5% to about 50% of CRM₁₉₇ lysines arecovalently linked to the saccharide. In some embodiments, the CRM₁₉₇ maycomprise about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, or about 16 lysineresidues out of 39 covalently linked to the saccharide.

In frequent embodiments, the carrier protein is covalently conjugated toan eTEC spacer through an amide linkage to one or more ε-amino groups oflysine residues on the carrier protein. In some such embodiments, thecarrier protein comprises 2 to 20 lysine residues covalently conjugatedto the saccharide. In other such embodiments, the carrier proteincomprises 4 to 16 lysine residues covalently conjugated to thesaccharide.

The serotype 33F glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide to carrier protein ratio(w/w) is between 0.2 and 4.0 (e.g., about 0.2, about 0.3, about 0.4,about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7,about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0,about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about3.7, about 3.8, about 3.9 or about 4.0). In other embodiments, thesaccharide to carrier protein ratio (w/w) is between 1.0 and 2.5. Infurther embodiments, the saccharide to carrier protein ratio (w/w) isbetween 0.4 and 1.7. In some such embodiments, the carrier protein isCRM₁₉₇.

The frequency of attachment of the saccharide chain to a lysine on thecarrier protein is another parameter for characterizing the serotype 33Fglycoconjugates of the invention. For example, in some embodiments, atleast one covalent linkage between the carrier protein and thepolysaccharide occurs for every 4 saccharide repeat units of thepolysaccharide. In another embodiment, the covalent linkage between thecarrier protein and the polysaccharide occurs at least once in every 10saccharide repeat units of the polysaccharide. In another embodiment,the covalent linkage between the carrier protein and the polysaccharideoccurs at least once in every 15 saccharide repeat units of thepolysaccharide. In a further embodiment, the covalent linkage betweenthe carrier protein and the polysaccharide occurs at least once in every25 saccharide repeat units of the polysaccharide.

In frequent embodiments, the carrier protein is CRM₁₉₇ and the covalentlinkage via an eTEC spacer between the CRM₁₉₇ and the polysaccharideoccurs at least once in every 4, 10, 15 or 25 saccharide repeat units ofthe polysaccharide.

In other embodiments, the conjugate comprises at least one covalentlinkage between the carrier protein and saccharide for every 5 to 10saccharide repeat units; every 2 to 7 saccharide repeat units; every 3to 8 saccharide repeat units; every 4 to 9 saccharide repeat units;every 6 to 11 saccharide repeat units; every 7 to 12 saccharide repeatunits; every 8 to 13 saccharide repeat units; every 9 to 14 sacchariderepeat units; every 10 to 15 saccharide repeat units; every 2 to 6saccharide repeat units, every 3 to 7 saccharide repeat units; every 4to 8 saccharide repeat units; every 6 to 10 saccharide repeat units;every 7 to 11 saccharide repeat units; every 8 to 12 saccharide repeatunits; every 9 to 13 saccharide repeat units; every 10 to 14 sacchariderepeat units; every 10 to 20 saccharide repeat units; every 4 to 25saccharide repeat units or every 2 to 25 saccharide repeat units. Infrequent embodiments, the carrier protein is CRM₁₉₇.

In another embodiment, at least one linkage between carrier protein andsaccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units ofthe polysaccharide. In an embodiment, the carrier protein is CRM₁₉₇. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure.

An important consideration during conjugation is the development ofconditions that permit the retention of potentially sensitivenon-saccharide substituent functional groups of the individualcomponents, such as O-Acyl, phosphate or glycerol phosphate side chainsthat may form part of the saccharide epitope.

In one embodiment, the serotype 33F glycoconjugates of the inventioncomprise a saccharide which has a degree of O-acetylation between 10%and 100%. In some such embodiments, the saccharide has a degree ofO-acetylation between 50% and 100%. In other such embodiments, thesaccharide has a degree of O-acetylation between 75% and 100%. Infurther embodiments, the saccharide has a degree of O-acetylationgreater than or equal to 70% (≥70%).

In a preferred embodiment, the serotype 33F glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mMacetate per mM serotype 33F capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.5, 0.6 or 0.7 mMacetate per mM serotype 33F capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.6 mM acetate per mMserotype 33F capsular polysaccharide. In a preferred embodiment, theglycoconjugate comprises at least 0.7 mM acetate per mM serotype 33Fcapsular polysaccharide. In a preferred embodiment, the presence ofO-acetyl groups is determined by ion-HPLC analysis.

In a preferred embodiment, the ratio of mM acetate per mM serotype 33Fpolysaccharide in the glycoconjugate to mM acetate per mM serotype 33Fpolysaccharide in the isolated polysaccharide is at least 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferred embodiment, the ratioof mM acetate per mM serotype 33F polysaccharide in the glycoconjugateto mM acetate per mM serotype 33F polysaccharide in the isolatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 33F polysaccharide in the glycoconjugate tomM acetate per mM serotype 33F polysaccharide in the isolatedpolysaccharide is at least 0.9.

In a preferred embodiment, the ratio of mM acetate per mM serotype 33Fpolysaccharide in the glycoconjugate to mM acetate per mM serotype 33Fpolysaccharide in the activated polysaccharide is at least 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferred embodiment, the ratioof mM acetate per mM serotype 33F polysaccharide in the glycoconjugateto mM acetate per mM serotype 33F polysaccharide in the activatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 33F polysaccharide in the glycoconjugate tomM acetate per mM serotype 33F polysaccharide in the activatedpolysaccharide is at least 0.9.

The serotype 33F glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 33F glycoconjugates of the inventioncomprise less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or5% of free serotype 33F polysaccharide compared to the total amount ofserotype 33F polysaccharide. Preferably, serotype 33F the glycoconjugatecomprises less than 15% free saccharide, more preferably less than 10%free saccharide, and still more preferably, less than 5% of freesaccharide. In a preferred embodiment the serotype 33F glycoconjugatecomprises less than about 25% of free serotype 33F polysaccharidecompared to the total amount of serotype 33F polysaccharide. In apreferred embodiment the serotype 33F glycoconjugate comprises less thanabout 20% of free serotype 33F polysaccharide compared to the totalamount of serotype 33F polysaccharide. In a preferred embodiment theserotype 33F glycoconjugate comprises less than about 15% of freeserotype 33F polysaccharide compared to the total amount of serotype 33Fpolysaccharide.

In certain preferred embodiments, the invention provides a serotype 33Fglycoconjugate having one or more of the following features alone or incombination: the polysaccharide has a molecular weight of between 50 kDaand 2,000 kDa; the glycoconjugate has a molecular weight of between 500kDa to 10,000 KDa; the carrier protein comprises 2 to 20 lysine residuescovalently linked to the saccharide; the saccharide to carrier proteinratio (w/w) is between 0.2 and 4.0; the glycoconjugate comprises atleast one covalent linkage between the carrier protein and thepolysaccharide for every 4, 10, 15 or 25 saccharide repeat units of thepolysaccharide; the saccharide has a degree of O-acetylation between 75%and 100%; the conjugate comprises less than about 15% freepolysaccharide relative to total polysaccharide; the carrier protein isCRM₁₉₇.

The serotype 33F glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above. In an embodiment, atleast 15% of the serotype 33F glycoconjugates of the invention have aK_(d) below or equal to 0.3 in a CL-4B column. In an embodiment, atleast 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% ofthe serotype 33F glycoconjugates of the invention have a K_(d) below orequal to 0.3 in a CL-4B column. In a preferred embodiment, at least 35%of the serotype 33F glycoconjugates of the invention have a K_(d) belowor equal to 0.3 in a CL-4B column. In preferred embodiments, at least40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serotype 33Fglycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 60% of the serotype33F glycoconjugates of the invention have a K_(d) below or equal to 0.3in a CL-4B column. In a preferred embodiment, at least 70% of theserotype 33F glycoconjugates of the invention have a K_(d) below orequal to 0.3 in a CL-4B column.

In a preferred embodiment, between 40% and 90% of the serotype 33Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 50% and 90% of the serotype 33Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 65% and 80% of the serotype 33Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column.

1.3.4 Glycoconjugates from S. pneumoniae Serotype 15B

In an embodiment, the serotype 15B glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/29094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 15B glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps, (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit, (2) reduction of the activated polysaccharide and a carrierprotein to form a conjugate.

Preferably, before oxidation, sizing of the serotype 15B polysaccharideto a target molecular weight (MW) range is performed. Advantageously,the size of the purified serotype 15B polysaccharide is reduced whilepreserving critical features of the structure of the polysaccharide suchas for example the presence of O-acetyl groups. Preferably, the size ofthe purified serotype 15B polysaccharide is reduced by mechanicalhomogenization (see section 1.2.6 above).

The oxidation step may involve reaction with periodate. For the purposeof the present invention, the term “periodate” includes both periodateand periodic acid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁵⁻) and the various salts of periodate (e.g., sodiumperiodate and potassium periodate). In a preferred embodiment theperiodate used for the oxidation of serotype 15B capsular polysaccharideis metaperiodate. In a preferred embodiment the periodate used for theoxidation of serotype 15B capsular polysaccharide is sodiummetaperiodate.

In a preferred embodiment, the polysaccharide is reacted with 0.01 to10.0, 0.05 to 5.0, 0.1 to 1.0, 0.5 to 1.0, 0.7 to 0.8, 0.05 to 0.5, 0.1to 0.3 molar equivalents of oxidizing agent. In a preferred embodiment,the polysaccharide is reacted with about 0.1, 0.15, 0.2, 0.25, 0.3,0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95molar equivalents of oxidizing agent. In a preferred embodiment, thepolysaccharide is reacted with about 0.15 molar equivalents of oxidizingagent. In a preferred embodiment, the polysaccharide is reacted withabout 0.25 molar equivalents of oxidizing agent. In a preferredembodiment, the polysaccharide is reacted with about 0.5 molarequivalents of oxidizing agent. In a preferred embodiment, thepolysaccharide is reacted with about 0.6 molar equivalents of oxidizingagent. In a preferred embodiment, the polysaccharide is reacted withabout 0.7 molar equivalents of oxidizing agent.

In a preferred embodiment, the duration of the reaction is between 1hour and 50 hours, between 10 hours and 30 hours, between 15 hours and20 hours, between 15 hours and 17 hours or about 16 hours.

In a preferred embodiment, the temperature of the reaction is maintainedbetween 15° C. and 45° C., between 15° C. and 30° C., between 20° C. and25° C. In a preferred embodiment, the temperature of the reaction ismaintained at about 23° C.

In a preferred embodiment, the oxidation reaction is carried out in abuffer selected from sodium phosphate, potassium phosphate,2-(N-morpholino)ethanesulfonic acid (MES) or Bis-Tris. In a preferredembodiment, the buffer is potassium phosphate.

In a preferred embodiment, the buffer has a concentration of between 1mM and 500 mM, between 1 mM and 300 mM, or between 50 mM and 200 mM. Ina preferred embodiment the buffer has a concentration of about 100 mM.

In a preferred embodiment, the oxidation reaction is carried out at a pHbetween 4.0 and 8.0, between 5.0 and 7.0, or between 5.5 and 6.5. In apreferred embodiment, the pH is about 6.0.

In preferred embodiment, the activated serotype 15B capsularpolysaccharide is obtained by reacting 0.5 mg/mL to 5 mg/mL of isolatedserotype 15B capsular polysaccharide with 0.2 to 0.3 molar equivalentsof periodate at a temperature between 20° C. and 25° C.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide is purified. The activated serotype 15B capsularpolysaccharide is purified according to methods known to the man skilledin the art, such as gel permeation chromatography (GPC), dialysis orultrafiltration/diafiltration. For example, the activated capsularpolysaccharide is purified by concentration and diafiltration using anultrafiltration device.

In a preferred embodiment, the degree of oxidation of the activatedserotype 15B capsular polysaccharide is between 2 and 20, between 2 and15, between 2 and 10, between 2 and 5, between 5 and 20, between 5 and15, between 5 and 10, between 10 and 20, between 10 and 15, or between15 and 20. In a preferred embodiment the degree of oxidation of theactivated serotype 15B capsular polysaccharide is between 2 and 10,between 4 and 8, between 4 and 6, between 6 and 8, between 6 and 12,between 8 and 12, between 9 and 11, between 10 and 16, between 12 and16, between 14 and 18, between 16 and 20, between 16 and 18, or between18 and 20.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide has a molecular weight between 5 kDa and 500 kDa, between50 kDa and 500 kDa, between 50 kDa and 450 kDa, between 100 kDa and 400kDa, between 100 kDa and 350 kDa. In a preferred embodiment, theactivated serotype 15B capsular polysaccharide has a molecular weightbetween 100 kDa and 350 kDa. In a preferred embodiment, the activatedserotype 15B capsular polysaccharide has a molecular weight between 100kDa and 300 kDa. In a preferred embodiment, the activated serotype 15Bcapsular polysaccharide has a molecular weight between 100 kDa and 250kDa.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or0.8 mM acetate per mM of said serotype 15B capsular polysaccharide. In apreferred embodiment, the activated serotype 15B capsular polysaccharidecomprises at least 0.5, 0.6 or 0.7 mM acetate per mM of said serotype15B capsular polysaccharide. In a preferred embodiment, the activatedserotype 15B capsular polysaccharide comprises at least 0.6 mM acetateper mM of said serotype 15B capsular polysaccharide. In a preferredembodiment, the activated serotype 15B capsular polysaccharide comprisesat least 0.7 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or0.8 mM glycerol per mM of said serotype 15B capsular polysaccharide. Ina preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.5, 0.6 or 0.7 mM glycerol per mM ofsaid serotype 15B capsular polysaccharide. In a preferred embodiment,the activated serotype 15B capsular polysaccharide comprises at least0.6 mM glycerol per mM of said serotype 15B capsular polysaccharide. Ina preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.7 mM glycerol per mM of saidserotype 15B capsular polysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 250 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 250 kDa andcomprises at least 0.6 mM glycerol per mM of said serotype 15B capsularpolysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide comprises at least 0.6 mM acetate per mM of said serotype15B capsular polysaccharide and at least 0.6 mM glycerol per mM of saidserotype 15B capsular polysaccharide.

In a preferred embodiment, the activated serotype 15B capsularpolysaccharide has a molecular weight between 100 kDa and 250 kDa andcomprises at least 0.6 mM acetate per mM of said serotype 15B capsularpolysaccharide and at least 0.6 mM glycerol per mM of said serotype 15Bcapsular polysaccharide.

In an embodiment, the activated serotype 15B capsular polysaccharide islyophilized, optionally in the presence of saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. Thelyophilized activated capsular polysaccharide can then be compoundedwith a solution comprising the carrier protein.

In another embodiment, the activated serotype 15B capsularpolysaccharide is compounded with the carrier protein and lyophilizedoptionally in the presence of a saccharide. In a preferred embodiment,the saccharide is selected from sucrose, trehalose, raffinose,stachyose, melezitose, dextran, mannitol, lactitol and palatinit. In apreferred embodiment, the saccharide is sucrose. The co-lyophilizedpolysaccharide and carrier protein can then be resuspended in solutionand reacted with a reducing agent.

The activated serotype 15B capsular polysaccharide can be conjugated toa carrier protein by a process comprising the step of:

(a) compounding the activated serotype 15B capsular polysaccharide witha carrier protein, and

(b) reacting the compounded activated serotype 15B capsularpolysaccharide and carrier protein with a reducing agent to form aserotype 15B capsular polysaccharide-carrier protein conjugate.

The conjugation of activated serotype 15B capsular polysaccharide with aprotein carrier by reductive amination in dimethylsulfoxide (DMSO) issuitable to preserve the O-acetyl content of the polysaccharide ascompared for example to reductive amination in aqueous solution wherethe level of O-acetylation of the polysaccharide is significantlyreduced. In a preferred embodiment, step (a) and step (b) are carriedout in DMSO.

In a preferred embodiment, step (a) comprises dissolving lyophilizedserotype 15B capsular polysaccharide in a solution comprising a carrierprotein and DMSO. In a preferred embodiment, step (a) comprisesdissolving co-lyophilized serotype 15B capsular polysaccharide andcarrier protein in DMSO.

When steps (a) and (b) are carried out in aqueous solution, steps (a)and (b) are carried out in a buffer, preferably selected from PBS, MES,HEPES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO,POPSO, TEA, EPPS, Bicine or HEPB, at a pH between 6.0 and 8.5, between7.0 and 8.0 or between 7.0 and 7.5. In a preferred embodiment the bufferis PBS. In a preferred embodiment the pH is about 7.3.

In a preferred embodiment, the concentration of activated serotype 15Bcapsular polysaccharide in step (b) is between 0.1 mg/mL and 10 mg/mL,between 0.5 mg/mL and 5 mg/mL, or between 0.5 mg/mL and 2 mg/mL. In apreferred embodiment, the concentration of activated serotype 15Bcapsular polysaccharide in step (b) is about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 mg/mL.

In a preferred embodiment the initial input ratio (weight by weight) ofactivated serotype 15B capsular polysaccharide to carrier protein isbetween 5:1 and 0.1:1, between 2:1 and 0.1:1, between 2:1 and 1:1,between 1.5:1 and 1:1, between 0.1:1 and 1:1, between 0.3:1 and 1:1, orbetween 0.6:1 and 1:1.

In a preferred embodiment the initial input ratio of activated serotype15B capsular polysaccharide to carrier protein is about 0.6:1 to 1:1. Inanother preferred embodiment the initial input ratio of activatedserotype 15B capsular polysaccharide to carrier protein is about 0.6:1to 1.5:1. Such initial input ratio is particularly suitable to obtainlow levels of free polysaccharide in the glycoconjugate.

In a preferred embodiment the initial input ratio of activated serotype15B capsular polysaccharide to carrier protein is about 0.4:1, 0.5:1,0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1,1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2:1.

In an embodiment, the reducing agent is sodium cyanoborohydride, sodiumtriacetoxyborohydride, sodium or zinc borohydride in the presence ofBronsted or Lewis acids, amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMeiPrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane(PEMB). In a preferred embodiment, the reducing agent is sodiumcyanoborohydride. In a preferred embodiment, the reducing agent issodium 2-Picoline Borane.

In a preferred embodiment, the quantity of reducing agent used in step(b) is between about 0.1 and 10.0 molar equivalents, between 0.5 and 5.0molar equivalents, or between 1.0 and 2.0 molar equivalents. In apreferred embodiment, the quantity of reducing agent used in step (b) isabout 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 molarequivalents.

In a preferred embodiment, the duration of step (b) is between 1 hourand 60 hours, between 10 hours and 50 hours, between 40 hours and 50hours, or between 42 hours and 46 hours. In a preferred embodiment, theduration of step (b) is about 44 hours.

In a preferred embodiment, the temperature of the reaction in step (b)is maintained between 10° C. and 40° C., between 15° C. and 30° C. orbetween 20° C. and 26° C. In a preferred embodiment, the temperature ofthe reaction in step (b) is maintained at about 23° C.

In a preferred embodiment, the process for the preparation of aglycoconjugate comprising S. pneumoniae serotype 15B capsularpolysaccharide covalently linked to a carrier protein further comprisesa step (step (c)) of capping unreacted aldehyde (quenching) by additionof NaBH₄.

In a preferred embodiment, the quantity of NaBH₄ used in step (c) isbetween 0.1 and 10 molar equivalents, between 0.5 and 5.0 molarequivalents or between 1.0 and 3.0 molar equivalents. In a preferredembodiment, the quantity of NaBH₄ used in step (c) is about 2 molarequivalents.

In a preferred embodiment, the duration of step (c) is between 0.1 hoursand 10 hours, 0.5 hours and 5 hours, or between 2 hours and 4 hours. Ina preferred embodiment, the duration of step (c) is about 3 hours.

In a preferred embodiment, the temperature of the reaction in step (c)is maintained between 15° C. and 45° C., between 15° C. and 30° C. orbetween 20° C. and 26° C. In a preferred embodiment, the temperature ofthe reaction in step (c) is maintained at about 23° C.

In a preferred embodiment the yield of the conjugation step is greaterthan 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%. In a preferredembodiment the yield of the conjugation step (step b) is greater than60%. In a preferred embodiment the yield of the conjugation step (stepb) is greater than 70%. The yield is the amount of serotype 15Bpolysaccharide in the conjugate×100)/amount of activated polysaccharideused in the conjugation step.

In a preferred embodiment, the process for the preparation of aglycoconjugate comprising S. pneumoniae serotype 15B capsularpolysaccharide covalently linked to a carrier protein comprises thesteps of:

(a) sizing purified serotype 15B polysaccharide by high pressurehomogenization;

(b) reacting the sized serotype 15B polysaccharide with an oxidizingagent;

(c) compounding the activated serotype 15B polysaccharide with a carrierprotein;

(d) reacting the compounded activated serotype 15B polysaccharide andcarrier protein with a reducing agent to form a serotype 15Bpolysaccharide-carrier protein conjugate; and

(e) capping unreacted aldehyde (quenching) by addition of NaBH₄.

In a preferred embodiment the yield of the conjugation step (step d) ofthe above process is greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%or 90%. In a preferred embodiment the yield of the conjugation step(step d) is greater than 60%. In a preferred embodiment the yield of theconjugation step (step d) is greater than 70%.

The yield is the amount of serotype 15B polysaccharide in theconjugate×100)/amount of activated polysaccharide used in theconjugation step.

After conjugation of the serotype 15B capsular polysaccharide to thecarrier protein, the polysaccharide-protein conjugate can be purified(enriched with respect to the amount of polysaccharide-proteinconjugate) by a variety of techniques known to the skilled person. Thesetechniques include dialysis, concentration/diafiltration operations,tangential flow filtration, precipitation/elution, column chromatography(DEAE or hydrophobic interaction chromatography), and depth filtration.

In an embodiment the carrier protein is as defined at section 1.1. In anembodiment the carrier protein is selected in the group consisting of:DT (Diphtheria toxin), TT (tetanus toxoid), CRM₁₉₇, other DT mutants, PD(Haemophilus influenzae protein D), or immunologically functionalequivalents thereof. In an embodiment the carrier protein is CRM₁₉₇.

In some embodiments, the serotype 15B glycoconjugates of the presentinvention are conjugated to the carrier protein (e.g., CRM₁₉₇) andcomprise a saccharide having a molecular weight of between 5 kDa and1,500 kDa. In other such embodiments, the saccharide has a molecularweight of between 10 kDa and 1,500 kDa. In further such embodiments, thesaccharide has a molecular weight of between 50 kDa and 1,500 kDa;between 50 kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50kDa and 750 kDa; between 50 kDa and 500 kDa; between 50 kDa and 250 kDa;between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and500 kDa; between 100 kDa and 250 kDa; between 200 kDa and 1,500 kDa;between 200 kDa and 1,250 kDa; between 200 kDa and 1,000 kDa; between200 kDa and 750 kDa; or between 200 kDa and 500 kDa; or between 200 kDaand 400 kDa. Any whole number integer within any of the above ranges iscontemplated as an embodiment of the disclosure. In some embodiments,the serotype 15B glycoconjugate of the invention has a molecular weightof between 50 kDa and 20,000 kDa. In some embodiments, the serotype 15Bglycoconjugate of the invention has a molecular weight of between 1,000kDa and 20,000 kDa In a preferred embodiment, the serotype 15Bglycoconjugate of the invention has a molecular weight between 3,000 kDaand 20,000 kDa, between 5,000 kDa and 10,000 kDa, between 5,000 kDa and20,000 kDa, between 8,000 kDa and 20,000 kDa, between 8,000 kDa and16,000 kDa or between 10,000 kDa and 16,000 kDa.

In further embodiments, the serotype 15B glycoconjugate of the inventionhas a molecular weight of about 1,000 kDa, about 1,500 kDa, about 2,000kDa, about 2,500 kDa, about 3,000 kDa, about 3,500 kDa, about 4,000 kDa,about 4,500 kDa, about 5,000 kDa, about 5,500 kDa, about 6,000 kDa,about 6,500 kDa, about 7,000 kDa, about 7,500 kDa, about 8,000 kDa,about 8,500 kDa, about 9,000 kDa, about 9,500 kDa about 10,000 kDa,about 10,500 kDa, about 11,000 kDa, about 11,500 kDa, about 12,000 kDa,about 12,500 kDa, about 13,000 kDa, about 13,500 kDa, about 14,000 kDa,about 14,500 kDa, about 15,000 kDa, about 15,500 kDa, about 16,000 kDa,about 16,500 kDa, about 17,000 kDa, about 17,500 kDa, about 18,000 kDa,about 18,500 kDa about 19,000 kDa, about 19,500 kDa or about 20,000 kDa.

In further embodiments, the serotype 15B glycoconjugate of the inventionhas a molecular weight of between 1,000 kDa and 20,000 kDa; between1,000 kDa and 15,000 kDa; between 1,000 kDa and 10,000 kDa; between1,000 kDa and 7,500 kDa; between 1,000 kDa and 5,000 kDa; between 1,000kDa and 4,000 kDa; between 1,000 kDa and 3,000 kDa; between 2,000 kDaand 20,000 kDa; between 2,000 kDa and 15,000 kDa; between 2,000 kDa and12,500 kDa; between 2,000 kDa and 10,000 kDa; between 2,000 kDa and7,500 kDa; between 2,000 kDa and 6,000 kDa; between 2,000 kDa and 5,000kDa; between 2,000 kDa and 4,000 kDa; or between 2,000 kDa and 3,000kDa.

In further embodiments, the serotype 15B glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 15,000 kDa; between 3,000 kDa and 10,000 kDa; between3,000 kDa and 7,500 kDa; between 3,000 kDa and 5,000 kDa; between 3,000kDa and 4,000 kDa; between 4,000 kDa and 20,000 kDa; between 4,000 kDaand 15,000 kDa; between 4,000 kDa and 12,500 kDa; between 4,000 kDa and10,000 kDa; between 4,000 kDa and 7,500 kDa; between 4,000 kDa and 6,000kDa or between 4,000 kDa and 5,000 kDa. In further embodiments, theserotype 15B glycoconjugate of the invention has a molecular weight ofbetween 5,000 kDa and 20,000 kDa; between 5,000 kDa and 15,000 kDa;between 5,000 kDa and 10,000 kDa; between 5,000 kDa and 7,500 kDa;between 6,000 kDa and 20,000 kDa; between 6,000 kDa and 15,000 kDa;between 6,000 kDa and 12,500 kDa; between 6,000 kDa and 10,000 kDa orbetween 6,000 kDa and 7,500 kDa.

The molecular weight of the glycoconjugate is measured by SEC-MALLS. Anywhole number integer within any of the above ranges is contemplated asan embodiment of the disclosure. In an embodiment, said serotype 15Bglycoconjugates are prepared using reductive amination.

The serotype 15B glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In a preferred embodiment, the ratio (weight by weight) ofserotype 15B capsular polysaccharide to carrier protein in the conjugateis between 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7, about0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4,about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7,about 2.8, about 2.9 or about 3.0). In a preferred embodiment, the ratioof serotype 15B capsular polysaccharide to carrier protein in theconjugate is between 0.4 and 2. In a preferred embodiment, the ratio ofserotype 15B capsular polysaccharide to carrier protein in the conjugateis between 0.5 and 2.0, 0.5 and 1.5, 0.5 and 1.0, 1.0 and 1.5, 1.0 and2.0. In a preferred embodiment, the ratio of serotype 15B capsularpolysaccharide to carrier protein in the conjugate is between 0.7 and0.9.

The serotype 15B glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In a preferred embodiment, the serotype 15B glycoconjugate of theinvention comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or15% of free serotype 15B capsular polysaccharide compared to the totalamount of serotype 15B capsular polysaccharide. In a preferredembodiment the serotype 15B glycoconjugate of the invention comprisesless than about 25% of free serotype 15B capsular polysaccharidecompared to the total amount of serotype 15B capsular polysaccharide. Ina preferred embodiment the serotype 15B glycoconjugate of the inventioncomprises less than about 20% of free serotype 15B capsularpolysaccharide compared to the total amount of serotype 15B capsularpolysaccharide. In a preferred embodiment the serotype 15Bglycoconjugates of the invention comprises less than about 15% of freeserotype 15B capsular polysaccharide compared to the total amount ofserotype 15B capsular polysaccharide.

The serotype 15B glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above.

In a preferred embodiment, at least 20% of the serotype 15Bglycoconjugates of the invention have a Kd below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 30% of the immunogenicconjugate has a Kd below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40% of the serotype 15B glycoconjugatesof the invention have a K_(d) below or equal to 0.3 in a CL-4B column.In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, or 85% of the serotype 15 glycoconjugates of the invention have aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 60% of the serotype 15B glycoconjugates of theinvention have a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 70% of the serotype 15B glycoconjugatesof the invention have a K_(d) below or equal to 0.3 in a CL-4B column.

In a preferred embodiment, between 40% and 90% of the serotype 15Bglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 50% and 90% of the serotype 15Bglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 65% and 80% of the serotype 15Bglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column.

In a preferred embodiment, the serotype 15B glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mMacetate per mM serotype 15B capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.5, 0.6 or 0.7 mMacetate per mM serotype 15B capsular polysaccharide. In a preferredembodiment, the glycoconjugate comprises at least 0.6 mM acetate per mMserotype 15B capsular polysaccharide. In a preferred embodiment, theglycoconjugate comprises at least 0.7 mM acetate per mM serotype 15Bcapsular polysaccharide. In a preferred embodiment, the presence ofO-acetyl groups is determined by ion-HPLC analysis.

In a preferred embodiment, the ratio of mM acetate per mM serotype 15Bcapsular polysaccharide in the serotype 15B glycoconjugate to mM acetateper mM serotype 15B capsular polysaccharide in the isolatedpolysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype15B capsular polysaccharide in the serotype 15B glycoconjugate to mMacetate per mM serotype 15B capsular polysaccharide in the isolatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 15B capsular polysaccharide in the serotype15B glycoconjugate to mM acetate per mM serotype 15B capsularpolysaccharide in the isolated polysaccharide is at least 0.9. In apreferred embodiment, the presence of O-acetyl groups is determined byion-HPLC analysis.

In a preferred embodiment, the ratio of mM acetate per mM serotype 15Bcapsular polysaccharide in the serotype 15B glycoconjugate to mM acetateper mM serotype 15B capsular polysaccharide in the activatedpolysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype15B capsular polysaccharide in the serotype 15B glycoconjugate to mMacetate per mM serotype 15B capsular polysaccharide in the activatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 15B capsular polysaccharide in the serotype15B glycoconjugate to mM acetate per mM serotype 15B capsularpolysaccharide in the activated polysaccharide is at least 0.9. In apreferred embodiment, the presence of O-acetyl groups is determined byion-HPLC analysis.

In a preferred embodiment, the serotype 15B glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mMglycerol per mM serotype 15B capsular polysaccharide. In a preferredembodiment, the serotype 15B glycoconjugate of the invention comprisesat least 0.5, 0.6 or 0.7 mM glycerol per mM serotype 15B capsularpolysaccharide. In a preferred embodiment, the serotype 15Bglycoconjugate of the invention comprises at least 0.6 mM glycerol permM serotype 15B capsular polysaccharide. In a preferred embodiment, theserotype 15B glycoconjugate of the invention comprises at least 0.7 mMglycerol per mM serotype 15B capsular polysaccharide.

Another way to characterize the serotype 15B glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials.

In a preferred embodiment, the degree of conjugation of the serotype 15Bglycoconjugate of the invention is between 2 and 15, between 2 and 13,between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5,between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10,between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4,between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12,between 10 and 15 or between 10 and 12. In an embodiment, the degree ofconjugation of the serotype 15B glycoconjugate of the invention is about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14 or about 15. In a preferredembodiment, the degree of conjugation of the serotype 15B glycoconjugateof the invention is between 2 and 5.

1.3.5 Glycoconjugates from S. pneumoniae Serotype 12F

In the glycoconjugates from S. pneumoniae serotype 12F of the presentinvention, the saccharide is selected from the group consisting of apolysaccharide and an oligosaccharide, and the carrier protein isselected from any suitable carrier as described herein or known to thoseof skill in the art. In some preferred embodiments, the saccharide is apolysaccharide from serotype 12F S. pneumoniae.

In an embodiment, glycoconjugates from S. pneumoniae serotype 12F areprepared using CDAP. The polysaccharides are activated with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated polysaccharide is then coupled directly orvia a spacer (linker) group to an amino group on the carrier protein(preferably CRM₁₉₇). For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example using GMBS) or ahaloacetylated carrier protein (for example using iodoacetimide, SIB,SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, the cyanate ester(optionally made by CDAP chemistry) is coupled with hexane diamine oradipic acid dihydrazide (ADH) and the amino-derivatised saccharide isconjugated to the carrier protein (e.g., CRM₁₉₇) using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier.

Other techniques for conjugation use carbodiimides, hydrazides, activeesters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS,EDC, TSTU. Many are described in International Patent ApplicationPublication No. WO 98/42721. Conjugation may involve a carbonyl linkerwhich may be formed by reaction of a free hydroxyl group of thesaccharide with CDI (see Bethell et al. (1979) J. Biol. Chem.254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518) followedby reaction with a protein to form a carbamate linkage. This may involvereduction of the anomeric terminus to a primary hydroxyl group, optionalprotection/deprotection of the primary hydroxyl group, reaction of theprimary hydroxyl group with CDI to form a CDI carbamate intermediate andcoupling the CDI carbamate intermediate with an amino group on aprotein.

In an embodiment, capsular polysaccharides from serotypes 12F S.pneumoniae are conjugated to the carrier protein by reductive amination.Reductive amination involves two steps, (1) oxidation of thepolysaccharide to generate aldehyde functionalities from vicinal diolsin individual hexasaccharide unit, (2) reduction of the activatedpolysaccharide and a carrier protein to form a conjugate.

Before oxidation, the serotype 12F polysaccharide is optionallyhydrolized (sized). Mechanical or chemical hydrolysis maybe employed.Chemical hydrolysis maybe conducted using acetic acid.

In an embodiment, the oxidizing agent is periodate. The term “periodate”includes both periodate and periodic acid (see below).

In a preferred embodiment, the oxidizing agent is2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical andN-Chlorosuccinimide (NCS) as the cooxidant. In such embodiment, theglycoconjugates from S. pneumoniae serotype 12F are prepared using2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical to oxidizeprimary alcohols of the saccharide to aldehydes usingN-Chlorosuccinimide (NCS) as the cooxidant (hereinafter “TEMPO/NCSoxidation”), such as described at Example 7 and in WO 2014/097099.Therefore in one aspect, the glycoconjugates from S. pneumoniae serotype12F are obtainable by a method comprising the steps of: a) reacting a12F saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) andN-chlorosuccinimide (NCS) in an aqueous solvent to produce an activatedsaccharide; and b) reacting the activated saccharide with a carrierprotein comprising one or more amine groups (hereinafter“TEMPO/NCS-reductive amination”). In one aspect, the glycoconjugatesfrom S. pneumoniae serotype 12F are obtained by said method. In anembodiment, the degree of oxidation of the activated 12F saccharideranges from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to10, from 1 to 5, from 3 to 40, from 3 to 30, from 3 to 20, from 3 to 10,from 4 to 40, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30,from 5 to 25, from 5 to 20, from 5 to 10, from 6 to 50, from 6 to 40,from 6 to 30, from 6 to 20, from 6 to 15, from 6 to 14, from 6 to 13,from 6 to 12, from 6 to 11, from 6 to 10, from 7 to 40, from 7 to 30,from 7 to 20, from 7 to 15, from 7 to 14, from 7 to 13, from 7 to 12,from 7 to 11, from 7 to 10, from 8 to 40, from 8 to 30, from 8 to 20,from 8 to 15, from 8 to 14, from 8 to 13, from 8 to 13, from 8 to 12,from 8 to 11, from 8 to 10, from 9 to 40, from 9 to 30, from 9 to 20,from 9 to 15, from 10 to 40, from 10 to 30, from 10 to 20, or from 10 to15. In a further aspect, the degree of oxidation of the activatedsaccharide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, or 40. Preferably, the carrier protein is CRM₁₉₇.

In an embodiment, prior to step a), the 12F saccharide is hydrolyzed toa molecular weight ranging from 100 kDa to 400 kDa. For example, in oneaspect, the molecular weight ranges from 100 kDa to 350 kDa, from 100kDa to 300 kDa, from 100 kDa to 250 kDa, from 100 kDa to 200 kDa, from100 kDa to 150 kDa, from 200 kDa to 400 kDa, from 200 kDa to 350 kDa,from 200 kDa to 300 kDa, from 200 kDa to 250 kDa, from 300 kDa to 400kDa, or from 300 kDa to 350 kDa.

In a further aspect, the method further comprises the step of purifyingthe activated polysaccharide prior to step b). In a further aspect, themethods further comprise the step of adding a reducing agent followingstep b). In one aspect, the reducing agent is NaCNBH₃. In a furtheraspect, the methods further comprise the step of adding NaBH₄ followingthe addition of NaCNBH₃. In a further aspect, the method comprises apurification step following the addition of NaBH₄.

In another aspect, the present disclosure provides a glycoconjugate fromS. pneumoniae serotype 12F produced, or obtainable by any of the methodsdisclosed hereabove. For example, in one aspect the present disclosureprovides a glycoconguate from S. pneumoniae serotype 12F comprising asaccharide conjugated to a carrier protein that is produced orobtainable by the method comprising the steps of: a) reacting asaccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) andN-chlorosuccinimide (NCS) in an aqueous solvent to produce an activatedsaccharide; and b) reacting the activated saccharide with a carrierprotein comprising one or more amine groups.

In one embodiment, the glycoconjugate from S. pneumoniae serotype 12F ofthe present invention has a molecular weight of between about 50 kDa andabout 20,000 kDa. In another embodiment, the glycoconjugate has amolecular weight of between about 200 kDa and about 10,000 kDa. Inanother embodiment, the glycoconjugate from S. pneumoniae serotype 12Fhas a molecular weight of between about 500 kDa and about 5,000 kDa. Inone embodiment, the glycoconjugate from S. pneumoniae serotype 12F has amolecular weight of between about 1,000 kDa and about 3,000 kDa. Inother embodiments the glycoconjugate from S. pneumoniae serotype 12F hasa molecular weight of between about 600 kDa and about 2,800 kDa; betweenabout 700 kDa and about 2,700 kDa; between about 1,000 kDa and about2,000 kDa; between about 1,800 kDa and about 2,500 kDa; between about1,100 kDa and about 2,200 kDa; between about 1,900 kDa and about 2,700kDa; between about 1,200 kDa and about 2,400 kDa; between about 1,700kDa and about 2,600 kDa; between about 1,300 kDa and about 2,600 kDa;between about 1,600 kDa and about 3,000 kDa.

In further embodiments, the serotype 12F glycoconjugate of the inventionhas a molecular weight of between 1,000 kDa and 20,000 kDa; between1,000 kDa and 15,000 kDa; between 1,000 kDa and 10,000 kDa; between1,000 kDa and 7,500 kDa; between 1,000 kDa and 5,000 kDa; between 1,000kDa and 4,000 kDa; between 1,000 kDa and 3,000 kDa; between 2,000 kDaand 20,000 kDa; between 2,000 kDa and 15,000 kDa; between 2,000 kDa and12,500 kDa; between 2,000 kDa and 10,000 kDa; between 2,000 kDa and7,500 kDa; between 2,000 kDa and 6,000 kDa; between 2,000 kDa and 5,000kDa; between 2,000 kDa and 4,000 kDa; or between 2,000 kDa and 3,000kDa. Any whole number integer within any of the above ranges iscontemplated as an embodiment of the disclosure. In some suchembodiments, the carrier protein is CRM₁₉₇. In some such embodiments,the serotype 12F glycoconjugate is conjugated to the carrier protein byTEMPO/NCS-reductive amination.

Another way to characterize the serotype 12F glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide, which can becharacterized as a range of conjugated lysines (degree of conjugation).

In a preferred embodiment, the degree of conjugation of the serotype 12Fglycoconjugate of the invention is between 2 and 20, between 4 and 16,between 4 and 15, between 2 and 15, between 2 and 13, between 2 and 10,between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4,between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8,between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15,between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15or between 10 and 12. In an embodiment, the degree of conjugation of theserotype 12F glycoconjugate of the invention is about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19 or about 20.

The number of lysine residues in the carrier protein conjugated to thesaccharide may also be expressed as a molar ratio. For example, in aglycoconjugate where 4 to 15 lysine residues of CRM₁₉₇ are covalentlylinked to the saccharide, the molar ratio of conjugated lysines toCRM₁₉₇ in the glycoconjugate is between about 10:1 to about 40:1. In animmunogenic composition where 2 to 20 lysine residues of CRM₁₉₇ arecovalently linked to the saccharide, the molar ratio of conjugatedlysines to CRM₁₉₇ in the glycoconjugate is between about 5:1 and about50:1. In one embodiment, in the glycoconjugate from S. pneumoniaeserotype 12F of the present invention the molar ratio of conjugatedlysines to carrier protein is from about 10:1 to about 25:1. In somesuch embodiments, the carrier protein is CRM₁₉₇. In some embodiments,the CRM₁₉₇ may comprise about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16 lysine residues out of 39 covalently linked to the saccharide. Insome such embodiments, the serotype 12F glycoconjugate is conjugated tothe carrier protein by TEMPO/NCS-reductive amination.

In one embodiment, the saccharide to carrier protein ratio (w/w) isbetween 0.2 and 4 in the glycoconjugate from S. pneumoniae serotype 12F(e.g., about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7,about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0,about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3,about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9 orabout 4.0). In another embodiment, the saccharide to carrier proteinratio (w/w) is between 1.1 and 1.7 in the glycoconjugate from S.pneumoniae serotype 12F. In other embodiments, the saccharide to carrierprotein ratio (w/w) is between 0.8 and 1.8 (e.g., about 0.8, about 0.9,about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about1.6, about 1.7 or about 1.8). In some such embodiments, the carrierprotein is CRM₁₉₇. In some such embodiments, the carrier protein isCRM₁₉₇. In some such embodiments, the serotype 12F glycoconjugate isconjugated to the carrier protein by TEMPO/NCS-reductive amination.

The frequency of attachment of the saccharide chain to a lysine on thecarrier protein is another parameter for characterizing the serotype 12Fglycoconjugates of the disclosure. For example, in one embodiment, thereis at least one covalent linkage between the carrier protein and thepolysaccharide for every 100 saccharide repeat units of thepolysaccharide. In one embodiment, there is at least one covalentlinkage between the carrier protein and the polysaccharide for every 50saccharide repeat units of the polysaccharide. In one embodiment, thereis at least one covalent linkage between the carrier protein and thepolysaccharide for every 25 saccharide repeat units of thepolysaccharide. In another embodiment, the covalent linkage between thecarrier protein and the polysaccharide occurs at least once in every 4saccharide repeat units of the polysaccharide. In another embodiment,the covalent linkage between the carrier protein and the polysaccharideoccurs at least once in every 10 saccharide repeat units of thepolysaccharide. In a further embodiment, the covalent linkage betweenthe carrier protein and the polysaccharide occurs at least once in every15 saccharide repeat units of the polysaccharide. In frequentembodiments, the carrier protein is CRM₁₉₇ and the covalent linkagebetween the CRM₁₉₇ and the polysaccharide occurs at least once in every4, 10, 15 or 25 saccharide repeat units of the polysaccharide.

In other embodiments, the conjugate comprises at least one covalentlinkage between the carrier protein and saccharide for every 5 to 10saccharide repeat units; every 2 to 7 saccharide repeat units; every 3to 8 saccharide repeat units; every 4 to 9 saccharide repeat units;every 6 to 11 saccharide repeat units; every 7 to 12 saccharide repeatunits; every 8 to 13 saccharide repeat units; every 9 to 14 sacchariderepeat units; every 10 to 15 saccharide repeat units; every 2 to 6saccharide repeat units, every 3 to 7 saccharide repeat units; every 4to 8 saccharide repeat units; every 6 to 10 saccharide repeat units;every 7 to 11 saccharide repeat units; every 8 to 12 saccharide repeatunits; every 9 to 13 saccharide repeat units; every 10 to 14 sacchariderepeat units; every 10 to 20 saccharide repeat units; every 4 to 25saccharide repeat units or every 2 to 25 saccharide repeat units. Infrequent embodiments, the carrier protein is CRM₁₉₇.

In another embodiment, at least one linkage between CRM₁₉₇ andsaccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units ofthe polysaccharide. In some such embodiments, the serotype 12Fglycoconjugate is conjugated to the carrier protein byTEMPO/NCS-reductive amination.

In one embodiment, the glycoconjugate from S. pneumoniae serotype 12F ofthe invention comprises at least one covalent linkage between thecarrier protein and the polysaccharide for every 25 saccharide repeatunits of the polysaccharide. In another embodiment, the covalent linkagebetween the carrier protein and the polysaccharide occurs at least oncein every 4 saccharide repeat units of the polysaccharide. In anotherembodiment, the covalent linkage between the carrier protein and thepolysaccharide occurs at least once in every 10 saccharide repeat unitsof the polysaccharide. In a further embodiment, the covalent linkagebetween the carrier protein and the polysaccharide occurs at least oncein every 15 saccharide repeat units of the polysaccharide. In some suchembodiments, the serotype 12F glycoconjugate is conjugated to thecarrier protein by TEMPO/NCS-reductive amination.

The serotype 12F glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 12F glycoconjugates of the inventioncomprise less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or5% of free serotype 12F polysaccharide compared to the total amount ofserotype 12F polysaccharide. In one embodiment, the glycoconjugate fromS. pneumoniae serotype 12F comprises less than about 50% of freeserotype 12F polysaccharide compared to the total amount of serotype 12Fpolysaccharide. In one embodiment, the glycoconjugate from S. pneumoniaeserotype 12F comprises less than about 45% of free serotype 12Fpolysaccharide compared to the total amount of serotype 12Fpolysaccharide. In another embodiment, the glycoconjugate comprises lessthan about 30% of free serotype 12F polysaccharide compared to the totalamount of serotype 12F polysaccharide. In another embodiment, theglycoconjugate from S. pneumoniae serotype 12F comprises less than about20% of free serotype 12F polysaccharide compared to the total amount ofserotype 12F polysaccharide. In a further embodiment, the glycoconjugatecomprises less than about 10% of free serotype 12F polysaccharidecompared to the total amount of serotype 12F polysaccharide. In anotherembodiment, the glycoconjugate from S. pneumoniae serotype 12F comprisesless than about 5% of free serotype 12F polysaccharide compared to thetotal amount of serotype 12F polysaccharide. In some such embodiments,the serotype 12F glycoconjugate is conjugated to the carrier protein byTEMPO/NCS-reductive amination.

In some embodiments, the serotype 12F glycoconjugate of the presentinvention comprises a saccharide having a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 2,000 kDa. In further suchembodiments, the saccharide has a molecular weight of between 50 kDa and1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa;between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDaand 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa; or between 200 kDa and 400 kDa. In some suchembodiments, the serotype 12F glycoconjugate is conjugated to thecarrier protein by TEMPO/NCS-reductive amination.

The serotype 12F glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above. In a preferredembodiment, at least 35% of the serotype 12F glycoconjugates of theinvention have a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, or 85% of the serotype 12F glycoconjugates of the invention have aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 60% of the serotype 12F glycoconjugates of theinvention have a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 70% of the serotype 12F glycoconjugatesof the invention have a K_(d) below or equal to 0.3 in a CL-4B column.

In a preferred embodiment, between 40% and 90% of the serotype 12Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 50% and 90% of the serotype 12Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 65% and 80% of the serotype 12Fglycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column.

1.3.6 Glycoconjugates from S. pneumoniae Serotype 10A

In an embodiment, the serotype 10A glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/29094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(See Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 10A glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps, (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit, (2) reduction of the activated polysaccharide and a carrierprotein to form a conjugate.

Before oxidation, the serotype 10A polysaccharide is optionallyhydrolized (sized). Mechanical or chemical hydrolysis maybe employed.Chemical hydrolysis maybe conducted using acetic acid.

In an embodiment, serotype polysaccharide is activated (oxidized) by aprocess comprising the step of:

(a) reacting isolated serotype 10A polysaccharide with an oxidizingagent;

(b) quenching the oxidation reaction by addition of a quenching agentresulting in an activated serotype 10A polysaccharide.

In a preferred embodiment, the oxidizing agent is periodate. For thepurpose of the present invention, the term “periodate” includes bothperiodate and periodic acid, the term also includes both metaperiodate(IO₄ ⁻) and orthoperiodate (IO₆ ⁵⁻) and the various salts of periodate(e.g., sodium periodate and potassium periodate). In a preferredembodiment, the oxidizing agent is sodium periodate. In a preferredembodiment, the periodate used for the oxidation of serotype 10Apolysaccharide is metaperiodate. In a preferred embodiment the periodateused for the oxidation of serotype 10A polysaccharide is sodiummetaperiodate.

In one embodiment, the quenching agent is selected from vicinal diols,1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid.

In one embodiment, the quenching agent is a 1,2-aminoalcohols of formula(I):

wherein R¹ is selected from H, methyl, ethyl, propyl or isopropyl.

In one embodiment, the quenching agent is selected from sodium andpotassium salts of sulfite, bisulfate, dithionite, metabisulfite,thiosulfate, phosphites, hypophosphites or phosphorous acid.

In one embodiment, the quenching agent is an amino acid. In suchembodiments, said amino acid may be selected from serine, threonine,cysteine, cystine, methionine, proline, hydroxyproline, tryptophan,tyrosine, and histidine.

In one embodiment, the quenching agent is a sulfite such as bisulfate,dithionite, metabisulfite, thiosulfate.

In one embodiment, the quenching agent is a compound comprising twovicinal hydroxyl groups (vicinal diols), i.e., two hydroxyl groupscovalently linked to two adjacent carbon atoms.

Preferably, the quenching agent is a compound of formula (II):

wherein R¹ and R² are each independently selected from H, methyl, ethyl,propyl or isopropyl.

In a preferred embodiment, the quenching agent is glycerol, ethyleneglycol, propan-1,2-diol, butan-1,2-diol or butan-2,3-diol, ascorbicacid. In a preferred embodiment, the quenching agent is butan-2,3-diol.

In preferred embodiment, the isolated serotype 10A polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 10A polysaccharide with periodate;

(b) quenching the oxidation reaction by addition of butan-2,3-diolresulting in an activated serotype 10A polysaccharide.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to an “activated polysaccharide”hereinafter.

In a preferred embodiment, the activated serotype 10A polysaccharide ispurified. The activated serotype 10A polysaccharide is purifiedaccording to methods known to the man skilled in the art, such as gelpermeation chromatography (GPC), dialysis orultrafiltration/diafiltration. For example, the activated 10Apolysaccharide is purified by concentration and diafiltration using anultrafiltration device.

In a preferred embodiment the degree of oxidation of the activatedserotype 10A polysaccharide is between 2 and 30, between 2 and 25,between 2 and 20, between 2 and 15, between 2 and 10, between 2 and 5,between 5 and 30, between 5 and 25, between 5 and 20, between 5 and 15,between 5 and 10, between 10 and 30, between 10 and 25, between 10 and20, between 10 and 15, between 15 and 30, between 15 and 25, between 15and 20, between 20 to 30, or between 20 to 25. In a preferred embodimentthe degree of oxidation of the activated serotype 10A polysaccharide isbetween 2 and 10, between 4 and 8, between 4 and 6, between 6 and 8,between 6 and 12, between 8 and 14, between 9 and 11, between 10 and 16,between 12 and 16, between 14 and 18, between 16 and 20, between 16 and18, between 18 and 22, or between 18 and 20.

In a preferred embodiment, the activated serotype 10A polysaccharide hasa molecular weight between 50 kDa and 400 kDa, between 50 kDa and 350kDa, between 50 kDa and 300 kDa, between 50 kDa and 250 kDa, between 50kDa and 200 kDa, between 100 kDa and 300 kDa, between 100 kDa and 250kDa or between 100 kDa and 200 kDa. In a preferred embodiment, theactivated serotype 10A polysaccharide has a molecular weight between 50kDa and 300 kDa. In a preferred embodiment, the activated serotype 10Apolysaccharide has a molecular weight between 100 kDa and 200 kDa. In apreferred embodiment, the activated serotype 10A polysaccharide has amolecular weight between 100 kDa and 200 kDa and a degree of oxidationbetween 5 and 20, between 5 and 15, between 8 and 14, between 8 and 12or between 9 and 11.

In a preferred embodiment, the activated serotype 10A polysaccharide hasa molecular weight between 100 kDa and 200 kDa and a degree of oxidationbetween 9 and 11.

The activated polysaccharide and/or the carrier protein may belyophilised (freeze-dried), either independently (discretelyophilization) or together (co-lyophilized).

In an embodiment, the activated serotype 10A polysaccharide islyophilized, optionally in the presence of saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. In oneembodiment, the lyophilized activated polysaccharide is then compoundedwith a solution comprising the carrier protein.

In another embodiment the activated polysaccharide and the carrierprotein are co-lyophilised. In such embodiments, the activated serotype10A polysaccharide is compounded with the carrier protein andlyophilized optionally in the presence of a saccharide. In a preferredembodiment, the saccharide is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the saccharide is sucrose. Theco-lyophilized polysaccharide and carrier protein can then beresuspended in solution and reacted with a reducing agent.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(reductive amination), using a reducing agent.

The activated serotype 10A polysaccharide can be conjugated to a carrierprotein by a process comprising the step of:

(c) compounding the activated serotype 10A polysaccharide with a carrierprotein; and

(d) reacting the compounded activated serotype 10A polysaccharide andcarrier protein with a reducing agent to form a serotype 10Apolysaccharide-carrier protein conjugate.

In an embodiment, the reduction reaction is carried out in aqueoussolvent, in another embodiment the reaction is carried out in aproticsolvent. In an embodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilised.

In an embodiment, the reducing agent is sodium cyanoborohydride, sodiumtriacetoxyborohydride, sodium or zinc borohydride in the presence ofBronsted or Lewis acids, amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMeiPrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane(PEMB). In a preferred embodiment, the reducing agent is sodiumcyanoborohydride.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄).

Following conjugation of serotype 10A polysaccharide to the carrierprotein, the glycoconjugate can be purified (enriched with respect tothe amount of polysaccharide-protein conjugate) by a variety oftechniques known to the skilled person. These techniques includedialysis, concentration/diafiltration operations, tangential flowfiltration precipitation/elution, column chromatography (DEAE orhydrophobic interaction chromatography), and depth filtration.

In some embodiments, the serotype 10A glycoconjugates of the presentinvention comprise a saccharide having a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 2,000 kDa. In further suchembodiments, the saccharide has a molecular weight of between 50 kDa and1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa;between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDaand 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa; or between 200 kDa and 400 kDa. In some suchembodiments, the serotype 10A glycoconjugates are prepared usingreductive amination.

In some embodiments, the serotype 10A glycoconjugate of the inventionhas a molecular weight of between 50 kDa and 20,000 kDa. In otherembodiments, the serotype 10A glycoconjugate has a molecular weight ofbetween 50 kDa and 15,000 kDa. In other embodiments, the serotype 10Aglycoconjugate has a molecular weight of between 500 kDa and 15,000 kDa,between 500 kDa and 10,000 kDa; between 2,000 kDa and 10,000 kDa; orbetween 3,000 kDa and 8,000 kDa. In other embodiments, the serotype 10Aglycoconjugate has a molecular weight of between 1,000 kDa and 10,000kDa. In other embodiments, the serotype 10A glycoconjugate has amolecular weight of between 1000 kDa and 8,000 kDa. In still otherembodiments, the serotype 10A glycoconjugate has a molecular weight ofbetween 2,000 kDa and 8,000 kDa or between 3,000 kDa and 7,000 kDa. Infurther embodiments, the serotype 10A glycoconjugate of the inventionhas a molecular weight of between 200 kDa and 20,000 kDa; between 200kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between 200 kDa and7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDa and 3,000 kDa;between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa; between 500 kDaand 10,000 kDa; between 500 kDa and 7,500 kDa; between 500 kDa and 6,000kDa; between 500 kDa and 5,000 kDa; between 500 kDa and 4,000 kDa;between 500 kDa and 3,000 kDa; between 500 kDa and 2,000 kDa; between500 kDa and 1,500 kDa; between 500 kDa and 1,000 kDa; between 750 kDaand 20,000 kDa; between 750 kDa and 15,000 kDa; between 750 kDa and12,500 kDa; between 750 kDa and 10,000 kDa; between 750 kDa and 7,500kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000 kDa;between 750 kDa and 4,000 kDa; between 750 kDa and 3,000 kDa; between750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between 1,000 kDaand 15,000 kDa; between 1,000 kDa and 12,500 kDa; between 1,000 kDa and10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa;between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 15,000 kDa;between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa;between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa;between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa; orbetween 2,000 kDa and 3,000 kDa.

In further embodiments, the serotype 10A glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 15,000 kDa; between 3,000 kDa and 10,000 kDa; between3,000 kDa and 7,500 kDa; between 3,000 kDa and 5,000 kDa; between 4,000kDa and 20,000 kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDaand 12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and5,000 kDa. In further embodiments, the serotype 10A glycoconjugate ofthe invention has a molecular weight of between 5,000 kDa and 20,000kDa; between 5,000 kDa and 15,000 kDa; between 5,000 kDa and 10,000 kDaor between 5,000 kDa and 7,500 kDa. In further embodiments, the serotype10A glycoconjugate of the invention has a molecular weight of between6,000 kDa and 20,000 kDa; between 6,000 kDa and 15,000 kDa; between6,000 kDa and 10,000 kDa or between 6,000 kDa and 7,500 kDa. In furtherembodiments, the serotype 10A glycoconjugate of the invention has amolecular weight of between 7,000 kDa and 20,000 kDa; between 7,000 kDaand 15,000 kDa; between 7,000 kDa and 10,000 kDa or between 7,000 kDaand 8,000 kDa. In further embodiments, the serotype 10A glycoconjugateof the invention has a molecular weight of between 8,000 kDa and 20,000kDa; between 8,000 kDa and 15,000 kDa; or between 8,000 kDa and 10,000kDa.

Any whole number integer within any of the above ranges is contemplatedas an embodiment of the disclosure. The molecular weight of theglycoconjugate is measured by SEC-MALLS.

Another way to characterize the serotype 10A glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials.

In a preferred embodiment, the degree of conjugation of the serotype 10Aglycoconjugate is between 2 and 15, between 2 and 13, between 2 and 10,between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4,between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8,between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15,between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15or between 10 and 12. In a preferred embodiment, the degree ofconjugation of the serotype 10A glycoconjugate is between 6 and 8. In apreferred embodiment, the carrier protein is CRM₁₉₇

The serotype 10A glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide to carrier protein ratio(w/w) is between 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7,about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0,about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about2.7, about 2.8, about 2.9 or about 3.0). In a preferred embodiment, theratio of serotype 10A saccharide to carrier protein in the conjugate isbetween 0.5 and 2.0, 0.5 and 1.5, 0.5 and 1.0, 1.0 and 1.5 or 1.0 and2.0. In a preferred embodiment, the ratio of serotype 10A polysaccharideto carrier protein in the conjugate is between 0.8 and 1.4. In apreferred embodiment, the ratio of serotype 10A capsular polysaccharideto carrier protein in the conjugate is between 0.8 and 1.2 (e.g., about0.8, about 0.9 about 1.0, about 1.1, or about 1.2). In some suchembodiments, the carrier protein is CRM₁₉₇.

The serotype 10A glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 10A glycoconjugates of the inventioncomprise less than about 50% free saccharide, less than about 45% freesaccharide, less than about 40% free saccharide, less than about 35%free saccharide, less than about 30% free saccharide, less than about25% free saccharide, less than about 20% free saccharide, less thanabout 15% free saccharide, less than about 10% free saccharide, or lessthan about 5% free saccharide relative to the total amount of 10Asaccharide. Preferably, serotype 10A the glycoconjugate comprises lessthan 15% free saccharide, more preferably less than 10% free saccharide,and still more preferably, less than 5% of free saccharide.

The serotype 10A glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above. In a preferredembodiment, at least 30% of the serotype 10A glycoconjugates of theinvention have a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40% of the serotype 10A glycoconjugatesof the invention have a K_(d) below or equal to 0.3 in a CL-4B column.In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, or 85% of the serotype 10A glycoconjugates of the invention have aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 60% of the serotype 10A glycoconjugates have aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, between 50% and 80% of the serotype 10A glycoconjugates ofthe invention have a K_(d) below or equal to 0.3 in a CL-4B column.

1.3.7 Glycoconjugates from S. pneumoniae Serotype 11A

In an embodiment, the serotype 11A glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/29094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979). Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 11A glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps, (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit, (2) reduction of the activated polysaccharide and a carrierprotein to form a conjugate.

Before oxidation, the serotype 11A polysaccharide is optionallyhydrolized to reduce its viscosity. Mechanical or chemical hydrolysismaybe employed. Chemical hydrolysis maybe conducted using acetic acid.Mechanical sizing maybe conducted using High Pressure HomogenizationShearing.

The oxidation step may involve reaction with periodate. For the purposeof the present invention, the term “periodate” includes both periodateand periodic acid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁵⁻) and the various salts of periodate (e.g., sodiumperiodate and potassium periodate). In an embodiment the capsularpolysaccharide from serotype 11A of S. pneumoniae is oxydized in thepresence of metaperiodate, preferably in the presence of sodiumperiodate (NaIO₄). In another embodiment the capsular polysaccharidefrom serotype 11A is oxydized in the presence of orthoperiodate,preferably in the presence of periodic acid.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to as “activated polysaccharide”here below. The activated polysaccharide maybe purified and lyophilised(freeze-dried).

The activated polysaccharide and the carrier protein may be lyophilized(freeze-dried), either independently (discrete lyophilization) ortogether (co-lyophilized). In one embodiment the activatedpolysaccharide and the carrier protein are co-lyophilized. In anotherembodiment the activated polysaccharide and the carrier protein arelyophilized independently.

In one embodiment the lyophilization takes place in the presence of anon-reducing sugar, possible non-reducing sugars include sucrose,trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitoland palatinit.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(reductive amination), using a reducing agent. Reducing agents which aresuitable include the cyanoborohydrides, such as sodium cyanoborohydride,borane-pyridine, or borohydride exchange resin. In one embodiment thereducing agent is sodium cyanoborohydride.

In an embodiment, the reduction reaction is carried out in aqueoussolvent, in another embodiment the reaction is carried out in aproticsolvent. In an embodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilised.

In one embodiment between 0.1 and 3.0, between 0.15 and 2.0, between 0.2and 2.0, or between 0.5 and 1.5 molar equivalents of

sodium cyanoborohydride is used in the reduction reaction. In oneembodiment about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.9 or 3.0 molar equivalents of sodium cyanoborohydride is used in thereduction reaction.

In one embodiment the reducing agent is sodium triacetoxyborohydride, ina further embodiment between 1.0 and 6.0 molar equivalents, between 2.0and 5.0 molar equivalents or about 3.0 molar equivalents of sodiumtriacetoxyborohydride is used in the reduction reaction.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent

is sodium borohydride (NaBH₄). In an embodiment capping is achieved bymixing the reduction reaction with between 0.5 and 5.0 molar equivalentsof NaBH4, for example about 1, 1.5, 2, 2.5 or 3 molar equivalents ofNaBH₄.

Following the conjugation (the reduction reaction and optionally thecapping), the glycoconjugates may be purified. The glycoconjugates maybepurified by diafiltration and/or ion exchange chromatography and/or

size exclusion chromatography. In an embodiment, the glycoconjugates arepurified by diafiltration or ion exchange chromatography or sizeexclusion chromatography.

In one embodiment the glycoconjugates are sterile filtered.

In some embodiments, the serotype 11A glycoconjugates of the presentinvention are conjugated to the carrier protein (e.g., CRM₁₉₇) andcomprise a saccharide having a molecular weight of between 10 kDa and2,000 kDa. In other such embodiments, the saccharide has a molecularweight of between 50 kDa and 2,000 kDa. In further such embodiments, thesaccharide has a molecular weight of between 50 kDa and 1,750 kDa;between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa; between 50kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500kDa; between 50 kDa and 400 kDa; between 50 kDa and 300 kDa; between 50kDa and 200 kDa; between 50 kDa and 100 kDa; between 100 kDa and 2,000kDa; between 100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa;between 100 kDa and 1,250 kDa; between 100 kDa and 1,000 kDa; between100 kDa and 750 kDa; between 100 kDa and 500 kDa; between 100 kDa and400 kDa between; 100 kDa and 300 kDa; between 100 kDa and 200 kDa;between 200 kDa and 2,000 kDa; between 200 kDa and 1,750 kDa; between200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa; between 200 kDaand 1,000 kDa; between 200 kDa and 750 kDa; or between 200 kDa and 500kDa; between 200 kDa and 400 kDa or between 200 kDa and 300 kDa.

In some embodiments, the serotype 11A glycoconjugate of the inventionhas a molecular weight of between 50 kDa and 20,000 kDa. In otherembodiments, the serotype 11A glycoconjugate has a molecular weight ofbetween 50 kDa and 15,000 kDa. In other embodiments, the serotype 11Aglycoconjugate has a molecular weight of between 500 kDa and 10,000 kDa.In other embodiments, the serotype 11A glycoconjugate has a molecularweight of between 200 kDa and 10,000 kDa. In still other embodiments,the serotype 11A glycoconjugate has a molecular weight of between 1,000kDa and 8,000 kDa or between 2,000 kDa and 8,000 kDa.

In further embodiments, the serotype 11A glycoconjugate of the inventionhas a molecular weight of between 200 kDa and 20,000 kDa; between 200kDa and 17,500 kDa; between 200 kDa and 15,000 kDa; between 200 kDa and10,000 kDa; between 200 kDa and 7,500 kDa; between 200 kDa and 5,000kDa; between 200 kDa and 3,000 kDa; between 200 kDa and 2,000 kDa;between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between500 kDa and 17,500 kDa; between 500 kDa and 15,000 kDa; between 500 kDaand 12,500 kDa; between 500 kDa and 10,000 kDa; between 500 kDa and7,500 kDa; between 500 kDa and 6,000 kDa; between 500 kDa and 5,000 kDa;between 500 kDa and 4,000 kDa; between 500 kDa and 3,000 kDa; between500 kDa and 2,000 kDa; between 500 kDa and 1,500 kDa; between 500 kDaand 1,000 kDa; between 700 kDa and 20,000 kDa; between 700 kDa and17,500 kDa; between 700 kDa and 15,000 kDa; between 700 kDa and 12,500kDa; between 700 kDa and 10,000 kDa; between 700 kDa and 7,500 kDa;between 700 kDa and 6,000 kDa; between 700 kDa and 5,000 kDa; between700 kDa and 4,500 kDa; between 700 kDa and 4,000 kDa; between 700 kDaand 3,500 kDa; between 700 kDa and 3,000 kDa; between 700 kDa and 2,000kDa; between 700 kDa and 1,500 kDa; between 1,000 kDa and 20,000 kDa;between 1,000 kDa and 17,500 kDa; between 1,000 kDa and 15,000 kDa;between 1,000 kDa and 12,500 kDa; between 1,000 kDa and 10,000 kDa;between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000 kDa;between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa;between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 20,000 kDa;between 2,000 kDa and 17,500 kDa; between 2,000 kDa and 15,000 kDa;between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa;between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa;between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa; orbetween 2,000 kDa and 3,000 kDa.

In further embodiments, the serotype 11A glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 17,500 kDa; between 3,000 kDa and 15,000 kDa; between3,000 kDa and 10,000 kDa; between 3,000 kDa and 7,500 kDa; between 3,000kDa and 5,000 kDa; between 4,000 kDa and 20,000 kDa; between 4,000 kDaand 17,500 kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDa and12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and5,000 kDa. In further embodiments, the serotype 11A glycoconjugate ofthe invention has a molecular weight of between 5,000 kDa and 20,000kDa; between 5,000 kDa and 17,500 kDa; between 5,000 kDa and 15,000 kDa;between 5,000 kDa and 10,000 kDa or between 5,000 kDa and 7,500 kDa.

In an embodiment, said serotype 11A glycoconjugates are prepared usingreductive amination.

In a preferred embodiment, the serotype 11A glycoconjugate of theinvention comprises at least 0.3, 0.5, 0.6, 1.0, 1.4, 1.8, 2.2, 2.6,3.0, 3.4, 3.8, 4.2, 4.6 or 5 mM acetate per mM serotype 11Apolysaccharide. In a preferred embodiment, the serotype 11Aglycoconjugate comprises at least 1.8, 2.2 or 2.6 mM acetate per mMserotype 11A polysaccharide. In an embodiment, the glycoconjugatecomprises at least 0.6 mM acetate per mM serotype 11A polysaccharide. Ina preferred embodiment, the serotype 11A glycoconjugate of the inventioncomprises at least 0.6, 1, 1.4, 1.8, 2.2, 2.6, 3, 3.4, 3.8, 4.2 or 4.6mM acetate per mM serotype 11A polysaccharide and less than about 5 mMacetate per mM serotype 11A polysaccharide. In an embodiment, theserotype 11A glycoconjugate of the invention comprises at least 0.6,1.0, 1.4, 1.8, 2.2, 2.6, or 3.0 mM acetate per mM serotype 11Apolysaccharide and less than about 3.4 mM acetate per mM serotype 11Apolysaccharide. In an embodiment, the serotype 11A glycoconjugate of theinvention comprises at least 0.6, 1, 1.4, 1.8, 2.2, 2.6, or about 3.0 mMacetate per mM serotype 11A polysaccharide and less than about 3.3 mMacetate per mM serotype 11A polysaccharide. Any of the above number iscontemplated as an embodiment of the disclosure.

In a preferred embodiment, the ratio of mM acetate per mM serotype 11Acapsular polysaccharide in the serotype 11A glycoconjugate to mM acetateper mM serotype 11A capsular polysaccharide in the isolatedpolysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype11A capsular polysaccharide in the serotype 11A glycoconjugate to mMacetate per mM serotype 11A capsular polysaccharide in the isolatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 11A capsular polysaccharide in the serotype11A glycoconjugate to mM acetate per mM serotype 11A capsularpolysaccharide in the isolated polysaccharide is at least 0.9. In apreferred embodiment, the presence of O-acetyl groups is determined byion-HPLC analysis.

In a preferred embodiment, the ratio of mM acetate per mM serotype 11Acapsular polysaccharide in the serotype 11A glycoconjugate to mM acetateper mM serotype 11A capsular polysaccharide in the activatedpolysaccharide is at least 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or0.95. In a preferred embodiment, the ratio of mM acetate per mM serotype11A capsular polysaccharide in the serotype 11A glycoconjugate to mMacetate per mM serotype 11A capsular polysaccharide in the activatedpolysaccharide is at least 0.7. In a preferred embodiment, the ratio ofmM acetate per mM serotype 11A capsular polysaccharide in the serotype11A glycoconjugate to mM acetate per mM serotype 11A capsularpolysaccharide in the activated polysaccharide is at least 0.9. In apreferred embodiment, the presence of O-acetyl groups is determined byion-HPLC analysis.

In a preferred embodiment, the serotype 11A glycoconjugate of theinvention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9or 1.0 mM glycerol per mM serotype 11A polysaccharide. In a preferredembodiment, the serotype 11A glycoconjugate comprises at least 0.2, 0.3or 0.4 mM glycerol per mM serotype 11A polysaccharide. In a preferredembodiment, the serotype 11A glycoconjugate of the invention comprisesat least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 mM glycerol permM serotype 11A polysaccharide and less than about 1.0 mM glycerol permM serotype 11A polysaccharide. In a preferred embodiment, the serotype11A glycoconjugate of the invention comprises at least 0.3, 0.4, 0.5,0.6, or 0.7 mM glycerol per mM serotype 11A polysaccharide and less thanabout 0.8 mM glycerol per mM serotype 11A polysaccharide. Any of theabove number is contemplated as an embodiment of the disclosure.

Another way to characterize the serotype 11A glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).

The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials.

In a preferred embodiment, the degree of conjugation of the serotype 11Aglycoconjugate of the invention is between 1 and 15, between 1 and 13,between 1 and 10, between 1 and 8, between 1 and 6, between 1 and 5,between 1 and 4, between 2 and 15, between 2 and 13, between 2 and 10,between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4,between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12,between 10 and 15 or between 10 and 12. In an embodiment, the degree ofconjugation of the serotype 11A glycoconjugate of the invention is about1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14 or about 15. In apreferred embodiment, the degree of conjugation of the serotype 11Aglycoconjugate of the invention is between 1 and 6 or between 2 and 5.In some such embodiments, the carrier protein is CRM₁₉₇.

The serotype 11A glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide to carrier protein ratio(w/w) is between 0.2 and 4 (e.g., about 0.2, about 0.3, about 0.4, about0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1,about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4,about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7,about 3.8, about 3.9 or about 4.0). In other embodiments, the saccharideto carrier protein ratio (w/w) is between 0.7 and 2.5, between 0.8 and2.0, between 0.7 and 2.0, between 0.8 and 1.5, between 0.7 and 1.5, 0.7and 1.4, between 0.8 and 1.4, between 0.7 and 1.45 or between 0.8 and1.45. In further embodiments, the saccharide to carrier protein ratio(w/w) is between 0.8 and 1.6 (e.g., about 0.8, about 0.9 about 1.0,about 1.1, about 1.2, about 1.3, about 1.4, about 1.5 or about 1.6). Insome such embodiments, the carrier protein is CRM₁₉₇. In an embodiment,said serotype 11A glycoconjugates are prepared using reductiveamination.

The serotype 11A glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 11A glycoconjugates of the inventioncomprise less than about 50% of free serotype 11A capsularpolysaccharide compared to the total amount of serotype 11A capsularpolysaccharide, less than about 45% free saccharide, less than about 40%free saccharide, less than about 35% free saccharide, less than about30% free saccharide, less than about 25% free saccharide, less thanabout 20% free saccharide, less than about 15% free saccharide, lessthan about 10% free saccharide, or less than about 5% of free serotype11A capsular polysaccharide compared to the total amount of serotype 11Acapsular polysaccharide. Preferably, serotype 11A the glycoconjugatecomprises less than 15% free saccharide, more preferably less than 10%free saccharide, and still more preferably, less than 5% of freesaccharide.

The serotype 11A glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate, as mentioned above. In a preferredembodiment, at least 30% of the serotype 11A glycoconjugates of theinvention has a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, or 85% of the serotype 11A glycoconjugates of the invention has aK_(d) below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 60% of the serotype 11A glycoconjugates of theinvention has a K_(d) below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 65% of the serotype 11A glycoconjugatesof the invention has a K_(d) below or equal to 0.3 in a CL-4B column.

1.3.8 Glycoconjugates from S. pneumoniae Serotype 8

In an embodiment, the serotype 8 glycoconjugates are obtained byactivating polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may be coupled directly or via a spacer (linker) group toan amino group on the carrier protein. For example, the spacer could becystamine or cysteamine to give a thiolated polysaccharide which couldbe coupled to the carrier via a thioether linkage obtained afterreaction with a maleimide-activated carrier protein (for example usingGMBS) or a haloacetylated carrier protein (for example usingiodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP). Preferably, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide(e.g., EDAC or EDC) chemistry via a carboxyl group on the proteincarrier. Such conjugates are described for example in WO 93/15760, WO95/08348 and WO 96/29094.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al.(1981) J. Chromatogr. 218:509-518) followed by reaction with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

In preferred embodiments, the serotype 8 glycoconjugates of theinvention are prepared using reductive amination. Reductive aminationinvolves two steps, (1) oxidation of the polysaccharide to generatealdehyde functionalities from vicinal diols in individual hexasaccharideunit, (2) reduction of the activated polysaccharide and a carrierprotein to form a conjugate.

Before oxidation, the serotype 8 polysaccharide is optionally hydrolizedto reduce its viscosity. Mechanical or chemical hydrolysis maybeemployed. Chemical hydrolysis maybe conducted using acetic acid.

The oxidation step may involve reaction with periodate. For the purposeof the present invention, the term “periodate” includes both periodateand periodic acid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁵⁻) and the various salts of periodate (e.g., sodiumperiodate and potassium periodate). In an embodiment the capsularpolysaccharide from serotype 8 of S. pneumoniae is oxydized in thepresence of metaperiodate, preferably in the presence of sodiumperiodate (NaIO₄). In another embodiment the capsular polysaccharidefrom serotype 8 is oxydized in the presence of orthoperiodate,preferably in the presence of periodic acid.

Following the oxidation step of the polysaccharide, the polysaccharideis said to be activated and is referred to as “activated polysaccharide”here below. The activated polysaccharide maybe purified and lyophilised(freeze-dried).

The activated polysaccharide and the carrier protein may be lyophilised(freeze-dried), either independently (discrete lyophilization) ortogether (co-lyophilized). In one embodiment the activatedpolysaccharide and the carrier protein are co-lyophilised. In anotherembodiment the activated polysaccharide and the carrier protein arelyophilised independently.

In one embodiment the lyophilisation takes place in the presence of anon-reducing sugar, possible non-reducing sugars include sucrose,trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitoland palatinit.

The second step of the conjugation process is the reduction of theactivated polysaccharide and a carrier protein to form a conjugate(reductive amination), using a reducing agent. Reducing agents which aresuitable include the cyanoborohydrides, such as sodium cyanoborohydride,borane-pyridine, or borohydride exchange resin. In one embodiment thereducing agent is sodium cyanoborohydride.

In an embodiment, the reduction reaction is carried out in aqueoussolvent, in another embodiment the reaction is carried out in aproticsolvent. In an embodiment, the reduction reaction is carried out in DMSO(dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO orDMF solvent may be used to reconstitute the activated polysaccharide andcarrier protein which has been lyophilised.

In one embodiment between 0.1 and 3.0, between 0.15 and 2.0, between 0.2and 1.0, or between 0.25 and 0.5 molar equivalents of sodiumcyanoborohydride is used in the reduction reaction. In one embodimentabout 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.9 or 3.0molar equivalents of sodium cyanoborohydride is used in the reductionreaction.

In one embodiment the reducing agent is sodium triacetoxyborohydride. Ina further embodiment between 1.0 and 6.0 molar equivalents, between 2.0and 5.0 molar equivalents or about 3.0 molar equivalents of sodiumtriacetoxyborohydride is used in the reduction reaction.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, these may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄). In an embodiment capping is achieved by mixing thereduction reaction with between 0.5 and 5.0 molar equivalents of NaBH₄,for example about 1.0, 1.5, 2.0, 2.5 or 3.0 molar equivalents of NaBH₄.

Following the conjugation (the reduction reaction and optionally thecapping), the glycoconjugates may be purified. The glycoconjugates maybepurified by diafiltration and/or ion exchange chromatography and/or sizeexclusion chromatography. In an embodiment, the glycoconjugates arepurified by diafiltration or ion exchange chromatography or sizeexclusion chromatography.

In one embodiment the glycoconjugates are sterile filtered.

In some embodiments, the serotype 8 glycoconjugates of the presentinvention are conjugated to the carrier protein (e.g., CRM₁₉₇) andcomprise a saccharide having a molecular weight of between 10 kDa and2,000 kDa. In other such embodiments, the saccharide has a molecularweight of between 50 kDa and 2,000 kDa. In further such embodiments, thesaccharide has a molecular weight of between 50 kDa and 1,750 kDa;between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa; between 50kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750 kDa;between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,750 kDa;between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa; between200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between 200 kDaand 500 kDa; or between 200 kDa and 400 kDa. In an embodiment, saidserotype 8 glycoconjugates are prepared using reductive amination.

In some embodiments, the serotype 8 glycoconjugate of the invention hasa molecular weight of between 50 kDa and 20,000 kDa. In otherembodiments, the serotype 8 glycoconjugate has a molecular weight ofbetween 50 kDa and 15,000 kDa. In other embodiments, the serotype 8glycoconjugate has a molecular weight of between 500 kDa and 10,000 kDa.In other embodiments, the serotype 8 glycoconjugate has a molecularweight of between 200 kDa and 10,000 kDa. In still other embodiments,the serotype 8 glycoconjugate has a molecular weight of between 1,000kDa and 8,000 kDa or between 2,000 kDa and 8,000 kDa.

In further embodiments, the serotype 8 glycoconjugate of the inventionhas a molecular weight of between 200 kDa and 20,000 kDa; between 200kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between 200 kDa and7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDa and 3,000 kDa;between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa; between 500 kDaand 10,000 kDa; between 500 kDa and 7,500 kDa; between 500 kDa and 6,000kDa; between 500 kDa and 5,000 kDa; between 500 kDa and 4,000 kDa;between 500 kDa and 3,000 kDa; between 500 kDa and 2,000 kDa; between500 kDa and 1,500 kDa; between 500 kDa and 1,000 kDa; between 750 kDaand 20,000 kDa; between 750 kDa and 15,000 kDa; between 750 kDa and12,500 kDa; between 750 kDa and 10,000 kDa; between 750 kDa and 7,500kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000 kDa;between 750 kDa and 4,000 kDa; between 750 kDa and 3,000 kDa; between750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between 1,000 kDaand 15,000 kDa; between 1,000 kDa and 12,500 kDa; between 1,000 kDa and10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa;between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 15,000 kDa;between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa;between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa;between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa; orbetween 2,000 kDa and 3,000 kDa.

In further embodiments, the serotype 8 glycoconjugate of the inventionhas a molecular weight of between 3,000 kDa and 20,000 kDa; between3,000 kDa and 15,000 kDa; between 3,000 kDa and 10,000 kDa; between3,000 kDa and 7,500 kDa; between 3,000 kDa and 5,000 kDa; between 4,000kDa and 20,000 kDa; between 4,000 kDa and 15,000 kDa; between 4,000 kDaand 12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 4,000 kDa and5,000 kDa. In further embodiments, the serotype 8 glycoconjugate of theinvention has a molecular weight of between 5,000 kDa and 20,000 kDa;between 5,000 kDa and 15,000 kDa; between 5,000 kDa and 10,000 kDa orbetween 5,000 kDa and 7,500 kDa. In further embodiments, the serotype 8glycoconjugate of the invention has a molecular weight of between 6,000kDa and 20,000 kDa; between 6,000 kDa and 15,000 kDa; between 6,000 kDaand 10,000 kDa or between 6,000 kDa and 7,500 kDa. In furtherembodiments, the serotype 8 glycoconjugate of the invention has amolecular weight of between 7,000 kDa and 20,000 kDa; between 7,000 kDaand 15,000 kDa; between 7,000 kDa and 10,000 kDa or between 7,000 kDaand 8,000 kDa. In further embodiments, the serotype 8 glycoconjugate ofthe invention has a molecular weight of between 8,000 kDa and 20,000kDa; between 8,000 kDa and 15,000 kDa; or between 8,000 kDa and 10,000kDa.

In an embodiment, said serotype 8 glycoconjugates are prepared usingreductive amination.

Another way to characterize the serotype 8 glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM₁₉₇) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).

The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art. Infrequent embodiments, the carrier protein is covalently conjugated toactivated polysaccharide through an amine linkage to one or more ε-aminogroups of lysine residues on the carrier protein. In some suchembodiments, the carrier protein comprises 2 to 20 lysine residuescovalently conjugated to the saccharide. In other such embodiments, thecarrier protein comprises 4 to 16 or 6 to 14 lysine residues covalentlyconjugated to the saccharide.

In a preferred embodiment, the degree of conjugation of the serotype 8glycoconjugate of the invention is between 2 and 20, between 2 and 15,between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6,between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13,between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5,between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15,between 8 and 12, between 10 and 15 or between 10 and 12. In anembodiment, the degree of conjugation of the serotype 8 glycoconjugateof the invention is about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 orabout 15. In a preferred embodiment, the degree of conjugation of theserotype 8 glycoconjugate of the invention is between 4 and 16 orbetween 6 and 14. In some such embodiments, the carrier protein isCRM₁₉₇.

In a preferred embodiment, the carrier protein comprises CRM₁₉₇, whichcontains 39 lysine residues. In some such embodiments, the CRM₁₉₇ maycomprise between 4 and 16 or between 6 and 14 lysine residues out of 39covalently linked to the saccharide.

Another way to express this parameter is that about 10% to about 41% orabout 15% to about 36% of CRM₁₉₇ lysines are covalently linked to thesaccharide. In another such embodiment, the CRM₁₉₇ may comprise 2 to 20lysine residues out of 39 covalently linked to the saccharide. Anotherway to express this parameter is that about 5% to about 50% of CRM₁₉₇lysines are covalently linked to the saccharide. In some suchembodiments, the CRM₁₉₇ may comprise about 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16 lysine residues out of 39 covalently linked to thesaccharide.

The serotype 8 glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide to carrier protein ratio(w/w) is between 0.2 and 4.0 (e.g., about 0.2, about 0.3, about 0.4,about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7,about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0,about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about3.7, about 3.8, about 3.9 or about 4.0). In other embodiments, thesaccharide to carrier protein ratio (w/w) is between 0.7 and 2.5. Infurther embodiments, the saccharide to carrier protein ratio (w/w) isbetween 0.8 and 1.5 (e.g., about 0.8, about 0.9 about 1.0, about 1.1,about 1.2, about 1.3, about 1.4 or about 1.5). In some such embodiments,the carrier protein is CRM₁₉₇. In an embodiment, said serotype 8glycoconjugates are prepared using reductive amination.

The serotype 8 glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be noncovalentlyassociated with (i.e., noncovalently bound to, adsorbed to, or entrappedin or with) the glycoconjugate.

In some embodiments, the serotype 8 glycoconjugates of the inventioncomprise less than about 50% free saccharide, less than about 45% freesaccharide, less than about 40% free saccharide, less than about 35%free saccharide, less than about 30% free saccharide, less than about25% free saccharide, less than about 20% free saccharide, less thanabout 15% free saccharide, less than about 10% free saccharide, or lessthan about 5% free saccharide relative to the total amount of serotype 8saccharide. Preferably, serotype 8 the glycoconjugate comprises lessthan 15% free saccharide, more preferably less than 10% free saccharide,and still more preferably, less than 5% of free saccharide.

The serotype 8 glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). Size exclusion chromatography media(CL-4B) can be used to determine the relative molecular sizedistribution of the conjugate. Size Exclusion Chromatography (SEC) isused in gravity fed columns to profile the molecular size distributionof conjugates. Large molecules excluded from the pores in the mediaelute more quickly than small molecules. Fraction collectors are used tocollect the column eluate. The fractions are tested colorimetrically bysaccharide assay. For the determination of K_(d), columns are calibratedto establish the fraction at which molecules are fully excluded (V₀),(K_(d)=0), and the fraction representing the maximum retention (V_(i)),(K_(d)=1). The fraction at which a specified sample attribute is reached(V_(e)), is related to K_(d) by the expression,K_(d)=(V_(e)−V₀)/(V_(i)−V₀).

In a preferred embodiment, at least 40% of the serotype 8glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serotype 8glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 60% of the serotype 8glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column. In a preferred embodiment, at least 70% of the serotype 8glycoconjugates of the invention have a K_(d) below or equal to 0.3 in aCL-4B column.

In a preferred embodiment, between 40% and 90% of the serotype 8glycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 50% and 90% of the serotype 8glycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column. Ina preferred embodiment, between 65% and 80% of the serotype 8glycoconjugates have a K_(d) below or equal to 0.3 in a CL-4B column.

1.4 Combinations of Glycoconjugates of the Invention

In an embodiment the immunogenic composition of the invention comprisesany of the glycoconjugates disclosed herein.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate selected from the group consisting of aglycoconjugate from S. pneumoniae serotype 15B (such as theglycoconjugates of section 1.3.4 above), a glycoconjugate from S.pneumoniae serotype 22F (such as the glycoconjugates of section 1.3.2above), a glycoconjugate from S. pneumoniae serotype 33F (such as theglycoconjugates of section 1.3.3 above), a glycoconjugate from S.pneumoniae serotype 12F (such as the glycoconjugates of section 1.3.5above), a glycoconjugate from S. pneumoniae serotype 10A (such as theglycoconjugates of section 1.3.6 above), a glycoconjugate from S.pneumoniae serotype 11A (such as the glycoconjugates of section 1.3.7above) and a glycoconjugate from S. pneumoniae serotype 8 (such as theglycoconjugates of section 1.3.8 above).

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate from S. pneumoniae serotype 15B, such as theglycoconjugate of section 1.3.4 above. In an embodiment the immunogeniccomposition of the invention comprises at least one glycoconjugate fromS. pneumoniae serotype 22F, such as the ones disclosed at section 1.3.2above. In an embodiment the immunogenic composition of the inventioncomprises at least one glycoconjugate from S. pneumoniae serotype 33Fsuch as the ones disclosed at section 1.3.3 above. In an embodiment theimmunogenic composition of the invention comprises at least oneglycoconjugate from S. pneumoniae serotype 12F such as the onesdisclosed at section 1.3.5 above. In an embodiment the immunogeniccomposition of the invention comprises at least one glycoconjugate fromS. pneumoniae serotype 10A such as the ones disclosed at section 1.3.6above. In an embodiment the immunogenic composition of the inventioncomprises at least one glycoconjugate from S. pneumoniae serotype 11Asuch as the ones disclosed at section 1.3.7 above. In an embodiment theimmunogenic composition of the invention comprises at least oneglycoconjugate from S. pneumoniae serotype 8 such as the ones disclosedat section 1.3.8 above.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the two S. pneumoniae serotypesselected from the group consisting of: 15B and 22F, 15B and 33F, 15B and12F, 15B and 10A, 15B and 11A, 15B and 8, 22F and 33F, 22F and 12F, 22Fand 10A, 22F and 11A, 22F and 8, 33F and 12F, 33F and 10A, 33F and 11A,33F and 8, 12F and 10A, 12F and 11A, 12F and 8, 10A and 11A, 10A and 8,and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the three following S. pneumoniaeserotypes:

15B and 22F and 33F,

15B and 22F and 12F,

15B and 22F and 10A,

15B and 22F and 11A,

15B and 22F and 8,

15B and 33F and 12F,

15B and 33F and 10A,

15B and 33F and 11A,

15B and 33F and 8,

15B and 12F and 10A,

15B and 12F and 11A,

15B and 12F and 8,

15B and 10A and 11A,

15B and 10A and 8,

15B and 11A and 8,

22F and 33F and 12F,

22F and 33F and 10A,

22F and 33F and 11A,

22F and 33F and 8,

22F and 12F and 10A,

22F and 12F and 11A,

22F and 12F and 8,

22F and 10A and 11A,

22F and 10A and 8,

22F and 11A and 8,

33F and 12F and 10A,

33F and 12F and 11A,

33F and 12F and 8,

33F and 10A and 11A,

33F and 10A and 8,

33F and 11A and 8,

12F and 10A and 11A,

12F and 10A and 8,

12F and 11A and 8, or

10A and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the four following S. pneumoniaeserotypes:

15B and 22F and 33F and 12F,

15B and 22F and 33F and 10A,

15B and 22F and 33F and 11A,

15B and 22F and 33F and 8,

15B and 22F and 12F and 10A,

15B and 22F and 12F and 11A,

15B and 22F and 12F and 8,

15B and 22F and 10A and 11A,

15B and 22F and 10A and 8,

15B and 22F and 11A and 8,

15B and 33F and 12F and 10A,

15B and 33F and 12F and 11A,

15B and 33F and 12F and 8,

15B and 33F and 10A and 11A,

15B and 33F and 10A and 8,

15B and 33F and 11A and 8,

15B and 12F and 10A and 11A,

15B and 12F and 10A and 8,

15B and 12F and 11A and 8,

15B and 10A and 11A and 8,

22F and 33F and 12F and 10A,

22F and 33F and 12F and 11A,

22F and 33F and 12F and 8,

22F and 33F and 10A and 11A,

22F and 33F and 10A and 8,

22F and 33F and 11A and 8,

22F and 12F and 10A and 11A,

22F and 12F and 10A and 8,

22F and 12F and 11A and 8,

22F and 10A and 11A and 8,

33F and 12F and 10A and 11A,

33F and 12F and 10A and 8,

33F and 12F and 11A and 8,

33F and 10A and 11A and 8 or

12F and 10A and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the five following S. pneumoniaeserotypes:

15B and 22F and 33F and 12F and 10A,

15B and 22F and 33F and 12F and 11A,

15B and 22F and 33F and 12F and 8,

15B and 22F and 33F and 10A and 11A,

15B and 22F and 33F and 10A and 8,

15B and 22F and 33F and 11A and 8,

15B and 22F and 12F and 10A and 11A,

15B and 22F and 12F and 10A and 8,

15B and 22F and 12F and 11A and 8,

15B and 22F and 10A and 11A and 8,

15B and 33F and 12F and 10A and 11A,

15B and 33F and 12F and 10A and 8,

15B and 33F and 12F and 11A and 8,

15B and 33F and 10A and 11A and 8,

15B and 12F and 10A and 11A and 8,

22F and 33F and 12F and 10A and 11A,

22F and 33F and 12F and 10A and 8,

22F and 33F and 12F and 11A and 8,

22F and 33F and 10A and 11A and 8,

22F and 12F and 10A and 11A and 8 or

33F and 12F and 10A and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the six following S. pneumoniaeserotypes:

15B and 22F and 33F and 12F and 10A and 11A,

15B and 22F and 33F and 12F and 10A and 8,

15B and 22F and 33F and 12F and 11A and 8,

15B and 22F and 33F and 10A and 11A and 8,

15B and 22F and 12F and 10A and 11A and 8,

15B and 33F and 12F and 10A and 11A and 8 or

22F and 33F and 12F and 10A and 11A and 8.

In an embodiment the immunogenic composition of the invention comprisesat least one glycoconjugate of each of the seven following S. pneumoniaeserotypes: 15B and 22F and 33F and 12F and 10A and 11A and 8.

In an embodiment the glycoconjugates from S. pneumoniae serotypes 15B,22F, 33F, 12F, 10A, 11A and/or 8 of any of the immunogenic compositiondefined in this section are as disclosed at sections 1.3.2 to 1.3.8above.

In an embodiment any of the immunogenic compositions above comprise inaddition glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14,18C, 19F and 23F (such as the glycoconjugates of section 1.3.1 above).

In an embodiment any of the immunogenic compositions above comprise inaddition glycoconjugates from S. pneumoniae serotypes 1, 5 and 7F (suchas the glycoconjugates of section 1.3.1 above).

In an embodiment any of the immunogenic compositions above comprise inaddition glycoconjugates from S. pneumoniae serotypes 6A and 19A (suchas the glycoconjugates of section 1.3.1 above).

In an embodiment any of the immunogenic compositions above comprise inaddition glycoconjugates from S. pneumoniae serotype 3 (such as theglycoconjugates of section 1.3.1 above).

Preferably, all the glycoconjugates of the above immunogeniccompositions are individually conjugated to the carrier protein.

In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 22F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 33F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 15B is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 12F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 10A is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 11A is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 8 is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and23F are conjugated to CRM₁₉₇. In an embodiment of any of the aboveimmunogenic compositions, the glycoconjugates from S. pneumoniaeserotypes 1, 5 and 7F are conjugated to CRM₁₉₇. In an embodiment of anyof the above immunogenic compositions, the glycoconjugates from S.pneumoniae serotypes 6A and 19A are conjugated to CRM₁₉₇. In anembodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotype 3 is conjugated to CRM₁₉₇.

In an embodiment, the glycoconjugates of any of the above immunogeniccompositions are all individually conjugated to CRM₁₉₇.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above immunogeniccompositions are individually conjugated to PD.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 18C ofany of the above immunogenic compositions is conjugated to TT.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 19F ofany of the above immunogenic compositions is conjugated to DT.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above immunogeniccompositions are individually conjugated to PD, the glycoconjugate fromS. pneumoniae serotype 18C is conjugated to TT and the glycoconjugatefrom S. pneumoniae serotype 19F is conjugated to DT.

In an embodiment the above immunogenic compositions comprise from 8 to20 different serotypes of S. pneumoniae. In one embodiment the aboveimmunogenic compositions comprise glycoconjugates from 12, 13, 14, 15,16, 17, 18, 19 or 20 different serotypes.

In one embodiment the above immunogenic compositions compriseglycoconjugates from 16 or 20 different serotypes.

In an embodiment the above immunogenic compositions are 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20-valent pneumococcal conjugatecompositions. In an embodiment the above immunogenic compositions are14, 15, 16, 17, 18 or 19-valent pneumococcal conjugate compositions. Inan embodiment the above immunogenic compositions are 16-valentpneumococcal conjugate compositions. In an embodiment the aboveimmunogenic compositions are 19-valent pneumococcal conjugatecompositions.

1. In an embodiment the immunogenic composition of the inventioncomprises at least one glycoconjugate from S. pneumoniae serotype 15B,such as the glycoconjugates of section 1.3.4 above.

2. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1 above, at least one glycoconjugate fromS. pneumoniae serotype 22F, such as the ones disclosed at section 1.3.2above.

3. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1 or 2 above, at least one glycoconjugatefrom S. pneumoniae serotype 33F such as the ones disclosed at section1.3.3 above.

4. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2 or 3 above, at least oneglycoconjugate from S. pneumoniae serotype 12F such as the onesdisclosed at section 1.3.5 above.

5. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3 or 4 above, at least oneglycoconjugate from S. pneumoniae serotype 10A such as the onesdisclosed at section 1.3.6 above.

6. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3, 4 or 5 above, at least oneglycoconjugate from S. pneumoniae serotype 11A such as the onesdisclosed at section 1.3.7 above.

7. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3, 4, 5 or 6 above, at least oneglycoconjugate from S. pneumoniae serotype 8 such as the ones disclosedat section 1.3.8 above.

8. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3, 4, 5, 6 or 7 aboveglycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and23F, such as the glycoconjugates of section 1.3.1 above.

9. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3, 4, 5, 6, 7 or 8 aboveglycoconjugates from S. pneumoniae serotypes 1, 5 and 7F such as theglycoconjugates of section 1.3.1 above.

10. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3, 4, 5, 6, 7, 8 or 9 aboveglycoconjugates from S. pneumoniae serotypes 6A and 19A such as theglycoconjugates of section 1.3.1 above.

11. In another embodiment the immunogenic composition of the inventioncomprises in addition to point 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aboveglycoconjugates from S. pneumoniae serotype 3 such as theglycoconjugates of section 1.3.1 above.

In an embodiment, the immunogenic composition of the invention comprisesglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In an embodiment, the immunogenic composition of the invention comprisesglycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V,14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In an embodiment, the immunogenic composition of the invention comprisesconjugated S. pneumoniae saccharides from serotypes 1, 3, 4, 5, 6A, 6B,7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In an embodiment, the immunogenic composition of the invention comprisesconjugated S. pneumoniae saccharides from serotypes 1, 4, 5, 6A, 6B, 7F,8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In an embodiment, the glycoconjugates of the immunogenic composition ofthe invention consist of glycoconjugates from S. pneumoniae serotypes 1,3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. In anembodiment, the glycoconjugates of the immunogenic composition of theinvention consist of glycoconjugates from serotypes 1, 4, 5, 6A, 6B, 7F,9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. In an embodiment, theglycoconjugates of the immunogenic composition of the invention consistof glycoconjugates from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A,11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. In an embodiment,the glycoconjugates of the immunogenic composition of the inventionconsist of glycoconjugates from 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

Preferably, all the glycoconjugates of the immunogenic composition ofthe invention (e.g., of any of points 1 to 11 above) are individuallyconjugated to the carrier protein.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of points 8 to 11 above areindividually conjugated to PD.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 18C ofany of points 8 to 11 above is conjugated to TT.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 19F ofany of points 8 to 11 above is conjugated to DT.

In an embodiment of any of points 8 to 11 above, the glycoconjugatesfrom S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F areindividually conjugated to PD, the glycoconjugate from S. pneumoniaeserotype 18C is conjugated to TT and the glycoconjugate from S.pneumoniae serotype 19F is conjugated to DT.

In an embodiment of any of points 1 to 11 above, the glycoconjugate fromS. pneumoniae serotype 22F is conjugated to CRM₁₉₇. In an embodiment ofany of points 2 to 11 above, the glycoconjugate from S. pneumoniaeserotype 33F is conjugated to CRM₁₉₇. In an embodiment of any of points3 to 11 above, the glycoconjugate from S. pneumoniae serotype 15B isconjugated to CRM₁₉₇. In an embodiment of any of points 4 to 11 above,the glycoconjugate from S. pneumoniae serotype 12F is conjugated toCRM₁₉₇. In an embodiment of any of points 5 to 11 above, theglycoconjugate from S. pneumoniae serotype 10A is conjugated to CRM₁₉₇.In an embodiment of any of points 6 to 11 above, the glycoconjugate fromS. pneumoniae serotype 11A is conjugated to CRM₁₉₇. In an embodiment ofany of points 7 to 11 above, the glycoconjugate from S. pneumoniaeserotype 8 is conjugated to CRM₁₉₇. In an embodiment of any of points 8to 11 above, the glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V,14, 18C, 19F and 23F are conjugated to CRM₁₉₇. In an embodiment of anyof points 9 to 11 above, the glycoconjugates from S. pneumoniaeserotypes 1, 5 and 7F are conjugated to CRM₁₉₇. In an embodiment of anyof points 10 to 11 above, the glycoconjugates from S. pneumoniaeserotypes 6A and 19A are conjugated to CRM₁₉₇. In an embodiment of point11 above, the glycoconjugate from S. pneumoniae serotype 3 is conjugatedto CRM₁₉₇.

In an embodiment, the glycoconjugates of immunogenic composition ofpoints 1 to 11 above are individually conjugated to CRM₁₉₇.

In an embodiment the immunogenic composition of the invention comprisesfrom 12 to 20 different serotypes of S. pneumoniae. In one embodimentthe immunogenic composition of the invention comprises glycoconjugatesfrom 12, 13, 14, 15, 16, 17, 18, 19 or 20 different serotypes. In oneembodiment the immunogenic composition of the invention comprisesglycoconjugates from 16 or 20 different serotypes.

In an embodiment the immunogenic composition of points 1 to 11 above isa 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-valent pneumococcalconjugate composition. In an embodiment the immunogenic composition ofpoints 1 to 11 above is a 15, 16, 17, 18 or 19-valent pneumococcalconjugate composition. In an embodiment the immunogenic composition ofpoints 1 to 11 above is a 16-valent pneumococcal conjugate composition.In an embodiment the immunogenic composition of points 1 to 11 above isa 19-valent pneumococcal conjugate composition.

After conjugation of the capsular polysaccharide to the carrier protein,the glycoconjugates are purified (enriched with respect to the amount ofpolysaccharide-protein conjugate) by a variety of techniques. Thesetechniques include concentration/diafiltration operations,precipitation/elution, column chromatography, and depth filtration (seefor example U.S. Patent App. Pub. No. 2007/0184072 or WO 2008/079653).After the individual glycoconjugates are purified, they are compoundedto formulate the immunogenic composition of the present invention.

1.5 Further Combinations of Glycoconjugates of the Invention

In an embodiment any of the immunogenic compositions defined at section1.4 above further comprise, at least one glycoconjugate from S.pneumoniae serotype 9V.

In an embodiment any of the immunogenic compositions defined at section1.4 above further comprise, at least one glycoconjugate of each of thetwo S. pneumoniae serotypes selected from the group consisting of: 9Vand 4, 9V and 6B, 9V and 14, 9V and 18C, 9V and 19F, 9V and 23F.

In an embodiment any of the immunogenic compositions defined at section1.4 above further comprise, at least one glycoconjugate of each of theseven following S. pneumoniae serotypes: 9V, 4, 6B, 14, 18C, 19F and23F.

In an embodiment any of the immunogenic compositions defined at section1.4 above further comprise, at least one glycoconjugate of each of theeight following S. pneumoniae serotypes:

9V and 1 and 4 and 6B and 14 and 18C and 19F and 23F,

9V and 4 and 5 and 6B and 14 and 18C and 19F and 23F, or

9V and 4 and 6B and 7F and 14 and 18C and 19F and 23F.

In an embodiment any of the immunogenic compositions defined at section1.4 above further comprise, at least one glycoconjugate of each of theten following S. pneumoniae serotypes: 9V, 1, 5, 4, 6B, 7F, 14, 18C, 19Fand 23F.

In an embodiment any of the immunogenic compositions defined at section1.4 above further comprise, at least one glycoconjugate of each of theeleven following S. pneumoniae serotypes:

9V and 1 and 4 and 5 and 6A and 6B and 7F and 14 and 18C and 19F and 23For

9V and 1 and 4 and 5 and 6B and 7F and 14 and 18C and 19A and 19F and23F.

In an embodiment any of the immunogenic compositions defined at section1.4 above further comprise, at least one glycoconjugate of each of thetwelve following S. pneumoniae serotypes: 9V, 1, 4, 5, 6A, 6B, 7F, 14,18C, 19A, 19F and 23F.

In an embodiment any of the immunogenic compositions defined at section1.4 above further comprise, at least one glycoconjugate of each of thethirteen following S. pneumoniae serotypes: 9V, 1, 3, 4, 5, 6A, 6B, 7F,14, 18C, 19A, 19F and 23F.

In an embodiment any of the immunogenic compositions defined at section1.4 above comprise in addition at least one glycoconjugate from S.pneumoniae serotype 2.

In an embodiment any of the immunogenic compositions defined at section1.4 above comprise in addition at least one glycoconjugate from S.pneumoniae serotype 17F.

In an embodiment any of the immunogenic compositions defined at section1.4 above comprise in addition at least one glycoconjugate from S.pneumoniae serotype 20.

In an embodiment any of the immunogenic compositions defined at section1.4 above comprise in addition at least one glycoconjugate from S.pneumoniae serotype 15C.

In an embodiment any of the immunogenic compositions defined at section1.4 above comprise in addition at least one glycoconjugate from S.pneumoniae serotype 9N.

Preferably, all the glycoconjugates of the above immunogeniccompositions are individually conjugated to the carrier protein.

In an embodiment of any of the above immunogenic compositions, theglycoconjugate from S. pneumoniae serotype 9V is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugates from S. pneumoniae serotypes 4, 6B, 14, 18C, 19F and 23Fare conjugated to CRM₁₉₇. In an embodiment of any of the aboveimmunogenic compositions, the glycoconjugates from S. pneumoniaeserotypes 1, 5 and 7F are conjugated to CRM₁₉₇. In an embodiment of anyof the above immunogenic compositions, the glycoconjugates from S.pneumoniae serotypes 6A and 19A are conjugated to CRM₁₉₇. In anembodiment of any of the above immunogenic compositions, theglycoconjugate from S. pneumoniae serotype 3 is conjugated to CRM₁₉₇. Inan embodiment of any of the above immunogenic compositions, theglycoconjugate from S. pneumoniae serotype 2 is conjugated to CRM₁₉₇. Inan embodiment of any of the above immunogenic compositions, theglycoconjugate from S. pneumoniae serotype 17F is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugate from S. pneumoniae serotype 20 is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugate from S. pneumoniae serotype 15C is conjugated to CRM₁₉₇.In an embodiment of any of the above immunogenic compositions, theglycoconjugate from S. pneumoniae serotype 9N is conjugated to CRM₁₉₇.

In an embodiment, the glycoconjugates of the above immunogeniccompositions are all individually conjugated to CRM₁₉₇.

In another embodiment, the glycoconjugate from S. pneumoniae serotype 9Vof any of the above immunogenic compositions is individually conjugatedto PD.

In an embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4,5, 6B, 7F, 9V, 14 and/or 23F of any of the above immunogeniccompositions are individually conjugated to PD.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 18C ofany of the above immunogenic compositions is conjugated to TT.

In an embodiment, the glycoconjugate from S. pneumoniae serotype 19F ofany of the above immunogenic compositions is conjugated to DT. In anembodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5,6B, 7F, 9V, 14 and/or 23F of any of the above immunogenic compositionsare individually

conjugated to PD, the glycoconjugate from S. pneumoniae serotype 18C isconjugated to TT and the glycoconjugate from S. pneumoniae serotype 19Fis conjugated to DT.

In an embodiment the above immunogenic compositions comprises from 7 to25 different serotypes of S. pneumoniae. In one embodiment the aboveimmunogenic compositions comprise glycoconjugates from 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 differentserotypes. In one embodiment the above immunogenic compositions compriseglycoconjugates from 16 or 20 different serotypes.

In an embodiment the above immunogenic compositions are 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20-valent pneumococcal conjugatecompositions. In an embodiment the above immunogenic compositions are14, 15, 16, 17, 18 or 19-valent pneumococcal conjugate compositions. Inan embodiment the above immunogenic compositions are 16-valentpneumococcal conjugate compositions. In an embodiment the aboveimmunogenic compositions are 19-valent pneumococcal conjugatecompositions. In an embodiment the above immunogenic compositions are20-valent pneumococcal conjugate compositions.

After conjugation of the capsular polysaccharide to the carrier protein,the glycoconjugates are purified (enriched with respect to the amount ofpolysaccharide-protein conjugate) by a variety of techniques. Thesetechniques include concentration/diafiltration operations,precipitation/elution, column chromatography, and depth filtration (seefor example U.S. Patent App. Pub. No. 2007/0184072 or WO 2008/079653.After the individual glycoconjugates are purified, they are compoundedto formulate the immunogenic composition of the present invention.

1.6 Particular Combinations of Glycoconjugates of the Invention

In an embodiment any of the immunogenic compositions defined at section1.4 or 1.5 above do not comprise capsular saccharide from S. pneumoniaeserotype 9N.

In an embodiment any of the immunogenic compositions defined at section1.4 or 1.5 above do not comprise capsular saccharide from S. pneumoniaeserotype 9A.

In an embodiment any of the immunogenic compositions defined at section1.4 or 1.5 above do not comprise capsular saccharide from S. pneumoniaeserotype 9L.

In an embodiment any of the immunogenic compositions defined at section1.4 or 1.5 above do not comprise capsular saccharide from S. pneumoniaeserotypes 9N and 9A.

In an embodiment any of the immunogenic compositions defined at section1.4 or 1.5 above do not comprise capsular saccharide from S. pneumoniaeserotypes 9N and 9L.

In an embodiment any of the immunogenic compositions defined at section1.4 or 1.5 above do not comprise capsular saccharide from S. pneumoniaeserotypes 9A and 9L.

In an embodiment any of the immunogenic compositions defined at section1.4 or 1.5 above do not comprise capsular saccharide from S. pneumoniaeserotypes 9N, 9A and 9L.

2 Dosage of the Immunogenic Compositions

The amount of glycoconjugate(s) in each dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccinees. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented.

2.1 Glycoconjugate Amount

The amount of a particular glycoconjugate in an immunogenic compositioncan be calculated based on total polysaccharide for that conjugate(conjugated and non-conjugated). For example, a glycoconjugate with 20%free polysaccharide will have about 80 μg of conjugated polysaccharideand about 20 μg of nonconjugated polysaccharide in a 100 μgpolysaccharide dose. The amount of glycoconjugate can vary dependingupon the pneumococcal serotype. The saccharide concentration can bedetermined by the uronic acid assay.

The “immunogenic amount” of the different polysaccharide components inthe immunogenic composition, may diverge and each may comprise about 1μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7μg, about 8 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg,about 90 μg, or about 100 μg of any particular polysaccharide antigen.

Generally, each dose will comprise 0.1 μg to 100 μg of polysaccharidefor a given serotype, particularly 0.5 μg to 20 μg, more particularly1.0 μg to 10 μg, and even more particularly 2.0 μg to 5.0 μg. Any wholenumber integer within any of the above ranges is contemplated as anembodiment of the disclosure.

In an embodiment, each dose will comprise about 1.0 μg, about 1.2 μg,about 1.4 μg, about 1.6 μg, about 1.8 μg, about 2.0 μg, about 2.2 μg,about 2.4 μg, about 2.6 μg, about 2.8 μg, about 3.0 μg, about 3.2 μg,about 3.4 μg, about 3.6 μg, about 3.8 μg, about 4.0 μg, about 4.2 μg,about 4.4 μg, about 4.6 μg, about 4.8 μg, about 5.0 μg, about 5.2 μg,about 5.4 μg, about 5.6 μg, about 5.8 μg or about 6.0 μg ofpolysaccharide for each particular glycoconjugate.

In an embodiment, each dose will comprise about 1.1 μg, about 1.2 μg,about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg,about 1.8 μg, about 1.9 μg, about 2.0 μg, about 2.1 μg, about 2.2 μg,about 2.3 μg, about 2.4 μg, about 2.5 μg, about 2.6 μg, about 2.7 μg,about 2.8 μg, about 2.9 μg, or about 3.0 μg μg of polysaccharide forglycoconjugates from S. pneumoniae serotype 1, 3, 4, 5, 6A, 7F, 8, 9V,10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and/or 33F.

In an embodiment, each dose will comprise about 1.1 μg, about 1.2 μg,about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg,about 1.8 μg, about 1.9 μg, about 2.0 μg, about 2.1 μg, about 2.2 μg,about 2.3 μg, about 2.4 μg, about 2.5 μg, about 2.6 μg, about 2.7 μg,about 2.8 μg, about 2.9 μg, or about 3.0 μg of polysaccharide forglycoconjugates from S. pneumoniae serotype 1, 4, 5, 6A, 7F, 8, 9V, 10A,11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and/or 33F.

In an embodiment, each dose will comprise about 2.0 μg, about 2.2 μg,about 2.4 μg, about 2.6 μg, about 2.8 μg, about 3.0 μg, about 3.2 μg,about 3.4 μg, about 3.6 μg, about 3.8 μg, about 4.0 μg, about 4.2 μg,about 4.4 μg, about 4.6 μg, about 4.8 μg, about 5.0, about 5.2 μg, about5.4 μg, about 5.6 μg, about 5.8 μg or about 6.0 μg of polysaccharide forglycoconjugates from S. pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 1.5 μg to about 3.0 μgof polysaccharide for each glycoconjugate from S. pneumoniae serotype 1,3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23Fand 33F, and about 3.0 μg to about 6.0 μg of polysaccharide forglycoconjugate from S. pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 2.0 μg to about 2.5 μgof polysaccharide for each glycoconjugate from S. pneumoniae serotype 1,3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23Fand 33F, and about 4.0 μg to about 4.8 μg of polysaccharide forglycoconjugate from S. pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 2.2 μg of polysaccharidefrom each glycoconjugate from S. pneumoniae serotype 1, 3, 4, 5, 6A, 7F,8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, andabout 4.4 μg of polysaccharide for glycoconjugate from S. pneumoniaeserotype 6B.

In an embodiment, each dose will comprise about 1.5 μg to about 3.0 μgof polysaccharide for each glycoconjugate from S. pneumoniae serotype 1,3, 4, 5, 6A, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about3 μg to about 6 μg of polysaccharide for glycoconjugate from S.pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 2.0 μg to about 2.5 μgof polysaccharide for each glycoconjugate from S. pneumoniae serotype 1,3, 4, 5, 6A, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about4.0 μg to about 4.8 μg of polysaccharide for glycoconjugate from S.pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 2.2 μg of polysaccharidefrom each glycoconjugate from S. pneumoniae serotype 1, 3, 4, 5, 6A, 7F,9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about 4.4 μg ofpolysaccharide for glycoconjugate from S. pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 1.5 μg to about 3.0 μgof polysaccharide for each glycoconjugate from S. pneumoniae serotype 1,4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and33F, and about 3.0 μg to about 6.0 μg of polysaccharide forglycoconjugate from S. pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 2.0 μg to about 2.5 μgof polysaccharide for each glycoconjugate from S. pneumoniae serotype 1,4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and33F, and about 4.0 μg to about 4.8 μg of polysaccharide forglycoconjugate from S. pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 2.2 μg of polysaccharidefrom each glycoconjugate from S. pneumoniae serotype 1, 4, 5, 6A, 7F, 8,9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about4.4 μg of polysaccharide for glycoconjugate from S. pneumoniae serotype6B.

In an embodiment, each dose will comprise about 1.5 μg to about 3.0 μgof polysaccharide for each glycoconjugate from S. pneumoniae serotype 1,4, 5, 6A, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about3.0 μg to about 6.0 μg of polysaccharide for glycoconjugate from S.pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 2.0 μg to about 2.5 μgof polysaccharide for each glycoconjugate from S. pneumoniae serotype 1,4, 5, 6A, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about4.0 μg to about 4.8 μg of polysaccharide for glycoconjugate from S.pneumoniae serotype 6B.

In an embodiment, each dose will comprise about 2.2 μg of polysaccharidefrom each glycoconjugate from S. pneumoniae serotype 1, 4, 5, 6A, 7F,9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, and about 4.4 μg ofpolysaccharide for glycoconjugate from S. pneumoniae serotype 6B.

2.2 Carrier Amount

Generally, each dose will comprise 10 μg to 150 μg of carrier protein,particularly 15 μg to 100 μg of carrier protein, more particularly 25 μgto 75 μg of carrier protein, and even more particularly 40 μg to 60 μgof carrier protein. In an embodiment, said carrier protein is CRM₁₉₇.

In an embodiment, each dose will comprise about 25 μg, about 26 μg,about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg,about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg,about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg,about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg,about 71 μg, about 72 μg, about 73 μg, about 74 μg or about 75 μg ofcarrier protein. In an embodiment, said carrier protein is CRM₁₉₇.

3 Further Antigens

Immunogenic compositions of the invention comprise conjugated S.pneumoniae saccharide antigens (glycoconjugates). They may also furtherinclude antigens from other pathogens, particularly from bacteria and/orviruses. Preferred further antigens are selected from: a diphtheriatoxoid (D), a tetanus toxoid (T), a pertussis antigen (P), which istypically acellular (Pa), a hepatitis B virus (HBV) surface antigen(HBsAg), a hepatitis A virus (HAV) antigen, a conjugated Haemophilusinfluenzae type b capsular saccharide (Hib), inactivated poliovirusvaccine (IPV).

In an embodiment, the immunogenic compositions of the invention compriseD-T-Pa. In an embodiment, the immunogenic compositions of the inventioncomprise D-T-Pa-Hib, D-T-Pa-IPV or D-T-Pa-HBsAg. In an embodiment, theimmunogenic compositions of the invention comprise D-T-Pa-HBsAg-IPV orD-T-Pa-HBsAg-Hib. In an embodiment, the immunogenic compositions of theinvention comprise D-T-Pa-HBsAg-IPV-Hib.

Pertussis antigens: Bordetella pertussis causes whooping cough.Pertussis antigens in vaccines are either cellular (whole cell, in theform of inactivated B. pertussis cells) or acellular. Preparation ofcellular pertussis antigens is well documented (e.g., it may be obtainedby heat inactivation of phase I culture of B. pertussis). Preferably,however, the invention uses acellular antigens. Where acellular antigensare used, it is preferred to use one, two or (preferably) three of thefollowing antigens: (1) detoxified pertussis toxin (pertussis toxoid, orPT); (2) filamentous hemagglutinin (FHA); (3) pertactin (also known asthe 69 kiloDalton outer membrane protein). FHA and pertactin may betreated with formaldehyde prior to use according to the invention. PT ispreferably detoxified by treatment with formaldehyde and/orglutaraldehyde. Acellular pertussis antigens are preferably adsorbedonto one or more aluminum salt adjuvants. As an alternative, they may beadded in an unadsorbed state. Where pertactin is added then it ispreferably already adsorbed onto an aluminum hydroxide adjuvant. PT andFHA may be adsorbed onto an aluminum hydroxide adjuvant or an aluminumphosphate. Adsorption of all of PT, FHA and pertactin to aluminumhydroxide is most preferred.

Inactivated poliovirus vaccine: Poliovirus causes poliomyelitis. Ratherthan use oral poliovirus vaccine, preferred embodiments of the inventionuse IPV. Prior to administration to patients, polioviruses must beinactivated, and this can be achieved by treatment with formaldehyde.Poliomyelitis can be caused by one of three types of poliovirus. Thethree types are similar and cause identical symptoms, but they areantigenically different and infection by one type does not protectagainst infection by others. It is therefore preferred to use threepoliovirus antigens in the invention: poliovirus Type 1 (e.g., Mahoneystrain), poliovirus Type 2 (e.g., MEF-1 strain), and poliovirus Type 3(e.g., Saukett strain). The viruses are preferably grown, purified andinactivated individually, and are then combined to give a bulk trivalentmixture for use with the invention.

Diphtheria toxoid: Corynebacterium diphtheriae causes diphtheria.Diphtheria toxin can be treated (e.g., using formalin or formaldehyde)to remove toxicity while retaining the ability to induce specificanti-toxin antibodies after injection. These diphtheria toxoids are usedin diphtheria vaccines. Preferred diphtheria toxoids are those preparedby formaldehyde treatment. The diphtheria toxoid can be obtained bygrowing C. diphtheriae in growth medium, followed by formaldehydetreatment, ultrafiltration and precipitation. The toxoided material maythen be treated by a process comprising sterile filtration and/ordialysis. The diphtheria toxoid is preferably adsorbed onto an aluminumhydroxide adjuvant.

Tetanus toxoid: Clostridium tetani causes tetanus. Tetanus toxin can betreated to give a protective toxoid. The toxoids are used in tetanusvaccines. Preferred tetanus toxoids are those prepared by formaldehydetreatment. The tetanus toxoid can be obtained by growing C. tetani ingrowth medium, followed by formaldehyde treatment, ultrafiltration andprecipitation. The material may then be treated by a process comprisingsterile filtration and/or dialysis.

Hepatitis A virus antigens: Hepatitis A virus (HAV) is one of the knownagents which causes viral hepatitis. A preferred HAV component is basedon inactivated virus, and inactivation can be achieved by formalintreatment.

Hepatitis B virus (HBV) is one of the known agents which causes viralhepatitis. The major component of the capsid is a protein known as HBVsurface antigen or, more commonly, HBsAg, which is typically a 226-aminoacid polypeptide with a molecular weight of ˜24 kDa. All existinghepatitis B vaccines contain HBsAg, and when this antigen isadministered to a normal vaccinee it stimulates the production ofanti-HBsAg antibodies which protect against HBV infection.

For vaccine manufacture, HBsAg has been made in two ways: purificationof the antigen in particulate form from the plasma of chronic hepatitisB carriers or expression of the protein by recombinant DNA methods(e.g., recombinant expression in yeast cells). Unlike native HBsAg(i.e., as in the plasma-purified product), yeast-expressed HBsAg isgenerally non-glycosylated, and this is the most preferred form of HBsAgfor use with the invention.

Conjugated Haemophilus influenzae type b antigens: Haemophilusinfluenzae type b (Hib) causes bacterial meningitis. Hib vaccines aretypically based on the capsular saccharide antigen, the preparation ofwhich is well documented. The Hib saccharide can be conjugated to acarrier protein in order to enhance its immunogenicity, especially inchildren. Typical carrier proteins are tetanus toxoid, diphtheriatoxoid, CRM₁₉₇ , H. influenzae protein D, and an outer membrane proteincomplex from serogroup B meningococcus. The saccharide moiety of theconjugate may comprise full-length polyribosylribitol phosphate (PRP) asprepared from Hib bacteria, and/or fragments of full-length PRP. Hibconjugates may or may not be adsorbed to an aluminum salt adjuvant.

In an embodiment the immunogenic compositions of the invention furtherinclude a conjugated N. meningitidis serogroup Y capsular saccharide(MenY), and/or a conjugated N. meningitidis serogroup C capsularsaccharide (MenC).

In an embodiment the immunogenic compositions of the invention furtherinclude a conjugated N. meningitidis serogroup A capsular saccharide(MenA), a conjugated N. meningitidis serogroup W135 capsular saccharide(MenW135), a conjugated N. meningitidis serogroup Y capsular saccharide(MenY), and/or a conjugated N. meningitidis serogroup C capsularsaccharide (MenC).

In an embodiment the immunogenic compositions of the invention furtherinclude a conjugated N. meningitidis serogroup W135 capsular saccharide(MenW135), a conjugated N. meningitidis serogroup Y capsular saccharide(MenY), and/or a conjugated N. meningitidis serogroup C capsularsaccharide (MenC).

4 Adjuvant(s)

In some embodiments, the immunogenic compositions disclosed herein mayfurther comprise at least one, two or three adjuvants. The term“adjuvant” refers to a compound or mixture that enhances the immuneresponse to an antigen. Antigens may act primarily as a delivery system,primarily as an immune modulator or have strong features of both.Suitable adjuvants include those suitable for use in mammals, includinghumans.

Examples of known suitable delivery-system type adjuvants that can beused in humans include, but are not limited to, alum (e.g., aluminumphosphate, aluminum sulfate or aluminum hydroxide), calcium phosphate,liposomes, oil-in-water emulsions such as MF59 (4.3% w/v squalene, 0.5%w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan trioleate (Span 85)),water-in-oil emulsions such as Montanide, andpoly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

In an embodiment, the immunogenic compositions disclosed herein comprisealuminum salts (alum) as adjuvant (e.g., aluminum phosphate, aluminumsulfate or aluminum hydroxide). In a preferred embodiment, theimmunogenic compositions disclosed herein comprise aluminum phosphate oraluminum hydroxide as adjuvant. In an embodiment, the immunogeniccompositions disclosed herein comprise from 0.1 mg/mL to 1 mg/mL or from0.2 mg/mL to 0.3 mg/mL of elemental aluminum in the form of aluminumphosphate. In an embodiment, the immunogenic compositions disclosedherein comprise about 0.25 mg/mL of elemental aluminum in the form ofaluminum phosphate. Examples of known suitable immune modulatory typeadjuvants that can be used in humans include, but are not limited to,saponin extracts from the bark of the Aquilla tree (QS21, Quil A), TLR4agonists such as MPL (Monophosphoryl Lipid A), 3DMPL (3-O-deacylatedMPL) or GLA-AQ, LT/CT mutants, cytokines such as the variousinterleukins (e.g., IL-2, IL-12) or GM-CSF, and the like.

Examples of known suitable immune modulatory type adjuvants with bothdelivery and immune modulatory features that can be used in humansinclude, but are not limited to, ISCOMS (see, e.g., Spölander et al.(1998) J. Leukocyte Biol. 64:713; WO 90/03184, WO 96/11711, WO 00/48630,WO 98/36772, WO 00/41720, WO 2006/134423 and WO 2007/026190) or GLA-EMwhich is a combination of a TLR4 agonist and an oil-in-water emulsion.

For veterinary applications including but not limited to animalexperimentation, one can use Complete Freund's Adjuvant (CFA), Freund'sIncomplete Adjuvant (IFA), Emulsigen,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

Further exemplary adjuvants to enhance effectiveness of the pneumococcalvaccines as disclosed herein include, but are not limited to: (1)oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (see below) orbacterial cell wall components), such as for example (a) SAF, containing10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, andthr-MDP either microfluidized into a submicron emulsion or vortexed togenerate a larger particle size emulsion, and (b) RIBI™ adjuvant system(RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2%Tween 80, and one or more bacterial cell wall components such asmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (DETOX™); (2) saponin adjuvants, suchas QS21, STIMULON™ (Cambridge Bioscience, Worcester, Mass.), ABISCO®(Isconova, Sweden), or ISCOMATRIX® (Commonwealth Serum Laboratories,Australia), may be used or particles generated therefrom such as ISCOMs(immunostimulating complexes), which ISCOMS may be devoid of additionaldetergent (e.g., WO 00/07621); (3) Complete Freund's Adjuvant (CFA) andIncomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins(e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g., WO 99/44636)),interferons (e.g., gamma interferon), macrophage colony stimulatingfactor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryllipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB2220211,EP0689454), optionally in the substantial absence of alum when used withpneumococcal saccharides (see, e.g., WO 00/56358); (6) combinations of3dMPL with, for example, QS21 and/or oil-in-water emulsions (see, e.g.,EP0835318, EP0735898, EP0761231); (7) a polyoxyethylene ether or apolyoxyethylene ester (see, e.g., WO 99/52549); (8) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (e.g., WO01/21207) or a polyoxyethylene alkyl ether or ester surfactant incombination with at least one additional non-ionic surfactant such as anoctoxynol (e.g., WO 01/21152); (9) a saponin and an immunostimulatoryoligonucleotide (e.g., a CpG oligonucleotide) (e.g., WO 00/62800); (10)an immunostimulant and a particle of metal salt (see, e.g., WO00/23105); (11) a saponin and an oil-in-water emulsion (e.g., WO99/11241); (12) a saponin (e.g., QS21)+3dMPL+IM2 (optionally+a sterol)(e.g., WO 98/57659); (13) other substances that act as immunostimulatingagents to enhance the efficacy of the composition. Muramyl peptidesinclude N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a CpG Oligonucleotide as adjuvant. A CpGoligonucleotide as used herein refers to an immunostimulatory CpGoligodeoxynucleotide (CpG ODN), and accordingly these terms are usedinterchangeably unless otherwise indicated. Immunostimulatory CpGoligodeoxynucleotides contain one or more immunostimulatory CpG motifsthat are unmethylated cytosine-guanine dinucleotides, optionally withincertain preferred base contexts. The methylation status of the CpGimmunostimulatory motif generally refers to the cytosine residue in thedinucleotide. An immunostimulatory oligonucleotide containing at leastone unmethylated CpG dinucleotide is an oligonucleotide which contains a5′ unmethylated cytosine linked by a phosphate bond to a 3′ guanine, andwhich activates the immune system through binding to Toll-like receptor9 (TLR-9). In another embodiment the immunostimulatory oligonucleotidemay contain one or more methylated CpG dinucleotides, which willactivate the immune system through TLR9 but not as strongly as if theCpG motif(s) was/were unmethylated. CpG immunostimulatoryoligonucleotides may comprise one or more palindromes that in turn mayencompass the CpG dinucleotide. CpG oligonucleotides have been describedin a number of issued patents, published patent applications, and otherpublications, including U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806;6,218,371; 6,239,116; and 6,339,068.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise any of the CpG Oligonucleotide described atpage 3, line 22, to page 12, line 36, of WO 2010/125480.

Different classes of CpG immunostimulatory oligonucleotides have beenidentified.

These are referred to as A, B, C and P class, and are described ingreater detail at page 3, line 22, to page 12, line 36, of WO2010/125480. Methods of the invention embrace the use of these differentclasses of CpG immunostimulatory oligonucleotides.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise an A class CpG oligonucleotide. Preferably,the “A class” CpG oligonucleotide of the invention has the followingnucleic acid sequence: 5′ GGGGACGACGTCGTGGGGGGG 3′ (SEQ ID NO: 1). Somenon-limiting examples of A-Class oligonucleotides include: 5′G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G 3′ (SEQ ID NO: 2); wherein “*”refers to a phosphorothioate bond and “_” refers to a phosphodiesterbond.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a B class CpG Oligonucleotide. In oneembodiment, the CpG oligonucleotide for use in the present invention isa B class CpG oligonucleotide represented by at least the formula:

5′ X₁X₂CGX₃X₄ 3′, wherein X1, X2, X3, and X4 are nucleotides. In oneembodiment, X₂ is adenine, guanine, or thymine. In another embodiment,X₃ is cytosine, adenine, or thymine.

The B class CpG oligonucleotide sequences of the invention are thosebroadly described above as well as disclosed in WO 96/02555, WO 98/18810and U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116and 6,339,068. Exemplary sequences include but are not limited to thosedisclosed in these latter applications and patents.

In an embodiment, the “B class” CpG oligonucleotide of the invention hasthe following nucleic acid sequence:

(SEQ ID NO: 3) 5′ TCGTCGTTTTTCGGTGCTTTT 3′, or (SEQ ID NO: 4)5′ TCGTCGTTTTTCGGTCGTTTT 3′, or (SEQ ID NO: 5)5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′, or (SEQ ID NO: 6)5′ TCGTCGTTTCGTCGTTTTGTCGTT 3′, or (SEQ ID NO: 7)5′ TCGTCGTTTTGTCGTTTTTTTCGA 3′.

In any of these sequences, all of the linkages may be allphosphorothioate bonds. In another embodiment, in any of thesesequences, one or more of the linkages may be phosphodiester, preferablybetween the “C” and the “G” of the CpG motif making a semi-soft CpGoligonucleotide. In any of these sequences, an ethyl-uridine or ahalogen may substitute for the 5′ T; examples of halogen substitutionsinclude but are not limited to bromo-uridine or iodo-uridinesubstitutions.

Some non-limiting examples of B-Class oligonucleotides include:

(SEQ ID NO: 8) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3′, or(SEQ ID NO: 9) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3′, or(SEQ ID NO: 10) 5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3′,or (SEQ ID NO: 11) 5′ T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T3′, or (SEQ ID NO: 12)5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A 3′.

wherein “*” refers to a phosphorothioate bond.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a C class CpG Oligonucleotide. In anembodiment, the “C class” CpG oligonucleotides of the invention have thefollowing nucleic acid sequence:

(SEQ ID NO: 13) 5′ TCGCGTCGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 14)5′ TCGTCGACGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 15)5′ TCGGACGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 16)5′ TCGGACGTTCGGCGCGCCG 3′, or (SEQ ID NO: 17)5′ TCGCGTCGTTCGGCGCGCCG 3′, or (SEQ ID NO: 18)5′ TCGACGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 19) 5′ TCGACGTTCGGCGCGCCG 3′,or (SEQ ID NO: 20) 5′ TCGCGTCGTTCGGCGCCG 3′, or (SEQ ID NO: 21)5′ TCGCGACGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 22)5′ TCGTCGTTTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 23)5′ TCGTCGTTTTCGGCGGCCGCCG 3′, or (SEQ ID NO: 24)5′ TCGTCGTTTTACGGCGCCGTGCCG 3′, or (SEQ ID NO: 25)5′ TCGTCGTTTTCGGCGCGCGCCGT 3′.

In any of these sequences, all of the linkages may be allphosphorothioate bonds. In another embodiment, in any of thesesequences, one or more of the linkages may be phosphodiester, preferablybetween the “C” and the “G” of the CpG motif making a semi-soft CpGoligonucleotide.

Some non-limiting examples of C-Class oligonucleotides include:

(SEQ ID NO: 26) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 27) 5′ T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 28) 5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 29) 5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′, or(SEQ ID NO: 30) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′, or(SEQ ID NO: 31) 5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 32) 5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′, or(SEQ ID NO: 33) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 3′, or(SEQ ID NO: 34) 5′ T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, or(SEQ ID NO: 35) 5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 3′, or(SEQ ID NO: 36) 5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G 3′, or(SEQ ID NO: 37) 5′ T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G 3′,or (SEQ ID NO: 38) 5′ T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3′ 

wherein “*” refers to a phosphorothioate bond and “_” refers to aphosphodiester bond. In any of these sequences, an ethyl-uridine or ahalogen may substitute for the 5′ T; examples of halogen substitutionsinclude but are not limited to bromo-uridine or iodo-uridinesubstitutions.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a P class CpG Oligonucleotide. In anembodiment, the CpG oligonucleotide for use in the present invention isa P class CpG oligonucleotide containing a 5′ TLR activation domain andat least two palindromic regions, one palindromic region being a 5′palindromic region of at least 6 nucleotides in length and connected toa 3′ palindromic region of at least 8 nucleotides in length eitherdirectly or through a spacer, wherein the oligonucleotide includes atleast one YpR dinucleotide. In an embodiment, said oligonucleotide isnot

T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G (SEQ ID NO: 27). In oneembodiment the P class CpG oligonucleotide includes at least oneunmethylated CpG dinucleotide. In another embodiment the TLR activationdomain is TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT,or TTTT. In yet another embodiment the TLR activation domain is withinthe 5′ palindromic region. In another embodiment the TLR activationdomain is immediately 5′ to the 5′ palindromic region. In an embodiment,the “P class” CpG oligonucleotides of the invention have the followingnucleic acid sequence:

(SEQ ID NO: 39) 5′ TCGTCGACGATCGGCGCGCGCCG 3′.

In said sequences, all of the linkages may be all phosphorothioatebonds. In another embodiment, one or more of the linkages may bephosphodiester, preferably between the “C” and the “G” of the CpG motifmaking a semi-soft CpG oligonucleotide. In any of these sequences, anethyl-uridine or a halogen may substitute for the 5′ T; examples ofhalogen substitutions include but are not limited to bromo-uridine oriodo-uridine substitutions.

A non-limiting example of P-Class oligonucleotides include:

(SEQ ID NO: 40) 5′ T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3′

wherein “*” refers to a phosphorothioate bond and “_” refers to aphosphodiester bond. In one embodiment the oligonucleotide includes atleast one phosphorothioate linkage. In another embodiment allinternucleotide linkages of the oligonucleotide are phosphorothioatelinkages. In another embodiment the oligonucleotide includes at leastone phosphodiester-like linkage. In another embodiment thephosphodiester-like linkage is a phosphodiester linkage. In anotherembodiment a lipophilic group is conjugated to the oligonucleotide. Inone embodiment the lipophilic group is cholesterol.

In an embodiment, all the internucleotide linkages of the CpGoligonucleotides disclosed herein are phosphodiester bonds (“soft”oligonucleotides, as described in WO 2007/026190). In anotherembodiment, CpG oligonucleotides of the invention are rendered resistantto degradation (e.g., are stabilized). A “stabilized oligonucleotide”refers to an oligonucleotide that is relatively resistant to in vivodegradation (e.g., via an exo- or endo-nuclease). Nucleic acidstabilization can be accomplished via backbone modifications.Oligonucleotides having phosphorothioate linkages provide maximalactivity and protect the oligonucleotide from degradation byintracellular exo- and endo-nucleases.

The immunostimulatory oligonucleotides may have a chimeric backbone,which have combinations of phosphodiester and phosphorothioate linkages.For purposes of the instant invention, a chimeric backbone refers to apartially stabilized backbone, wherein at least one internucleotidelinkage is phosphodiester or phosphodiester-like, and wherein at leastone other internucleotide linkage is a stabilized internucleotidelinkage, wherein the at least one phosphodiester or phosphodiester-likelinkage and the at least one stabilized linkage are different. When thephosphodiester linkage is preferentially located within the CpG motifsuch molecules are called “semi-soft” as described in WO 2007/026190.

Other modified oligonucleotides include combinations of phosphodiester,phosphorothioate, methylphosphonate, methylphosphorothioate,phosphorodithioate, and/or p-ethoxy linkages.

Mixed backbone modified ODN may be synthesized as described in WO2007/026190.

The size of the CpG oligonucleotide (i.e., the number of nucleotideresidues along the length of the oligonucleotide) also may contribute tothe stimulatory activity of the oligonucleotide. For facilitating uptakeinto cells, CpG oligonucleotide of the invention preferably have aminimum length of 6 nucleotide residues. Oligonucleotides of any sizegreater than 6 nucleotides (even many kb long) are capable of inducingan immune response if sufficient immunostimulatory motifs are present,because larger oligonucleotides are degraded inside cells. In certainembodiments, the CpG oligonucleotides are 6 to 100 nucleotides long,preferentially 8 to 30 nucleotides long. In important embodiments,nucleic acids and oligonucleotides of the invention are not plasmids orexpression vectors.

In an embodiment, the CpG oligonucleotide disclosed herein comprisesubstitutions or modifications, such as in the bases and/or sugars asdescribed at paragraphs 134 to 147 of WO 2007/026190.

In an embodiment, the CpG oligonucleotide of the present invention ischemically modified. Examples of chemical modifications are known to theskilled person and are described, for example in Uhlmann et al. (1990)Chem. Rev. 90:543; S. Agrawal, Ed., Humana Press, Totowa, USA 1993;Crooke et al. (1996) Annu. Rev. Pharmacol. Toxicol. 36:107-129; andHunziker et al. (1995) Mod. Synth. Methods 7:331-417. An oligonucleotideaccording to the invention may have one or more modifications, whereineach modification is located at a particular phosphodiesterinternucleoside bridge and/or at a particular β-D-ribose unit and/or ata particular natural nucleoside base position in comparison to anoligonucleotide of the same sequence which is composed of natural DNA orRNA.

In some embodiments of the invention, CpG-containing nucleic acids mightbe simply mixed with immunogenic carriers according to methods known tothose skilled in the art (see, e.g., WO 03/024480).

In a particular embodiment of the present invention, any of theimmunogenic compositions disclosed herein comprise from 2 μg to 100 mgof CpG oligonucleotide, preferably from 0.1 mg to 50 mg CpGoligonucleotide, preferably from 0.2 mg to 10 mg CpG oligonucleotide,preferably from 0.3 mg to 5 mg CpG oligonucleotide, preferably from 0.3mg to 5 mg CpG oligonucleotide, even preferably from 0.5 to 2 mg CpGoligonucleotide, even preferably from 0.75 to 1.5 mg CpGoligonucleotide. In a preferred embodiment, any of the immunogeniccomposition disclosed herein comprises about 1 mg CpG oligonucleotide.

5 Formulation

The immunogenic compositions of the invention may be formulated inliquid form (i.e., solutions or suspensions) or in a lyophilized form.Liquid formulations may advantageously be administered directly fromtheir packaged form and are thus ideal for injection without the needfor reconstitution in aqueous medium as otherwise required forlyophilized compositions of the invention.

Formulation of the immunogenic composition of the present invention canbe accomplished using art-recognized methods. For instance, theindividual pneumococcal conjugates can be formulated with aphysiologically acceptable vehicle to prepare the composition. Examplesof such vehicles include, but are not limited to, water, bufferedsaline, polyols (e.g., glycerol, propylene glycol, liquid polyethyleneglycol) and dextrose solutions.

The present disclosure provides an immunogenic composition comprisingany of combination of glycoconjugates disclosed herein and apharmaceutically acceptable excipient, carrier, or diluent.

In an embodiment, the immunogenic composition of the invention is inliquid form, preferably in aqueous liquid form.

Immunogenic compositions of the disclosure may comprise one or more of abuffer, a salt, a divalent cation, a non-ionic detergent, acryoprotectant such as a sugar, and an anti-oxidant such as a freeradical scavenger or chelating agent, or any multiple combinationsthereof.

In an embodiment, the immunogenic compositions of the invention comprisea buffer. In an embodiment, said buffer has a pKa of about 3.5 to about7.5. In some embodiments, the buffer is phosphate, succinate, histidineor citrate. In certain embodiments, the buffer is succinate at a finalconcentration of 1 mM to 10 mM. In one particular embodiment, the finalconcentration of the succinate buffer is about 5 mM.

In an embodiment, the immunogenic compositions of the invention comprisea salt. In some embodiments, the salt is selected from the groupsconsisting of magnesium chloride, potassium chloride, sodium chlorideand a combination thereof. In one particular embodiment, the salt issodium chloride. In one particular embodiment, the immunogeniccompositions of the invention comprise sodium chloride at 150 mM.

In an embodiment, the immunogenic compositions of the invention comprisea surfactant. In an embodiment, the surfactant is selected from thegroup consisting of polysorbate 20 (TWEEN™20), polysorbate 40(TWEEN™40), polysorbate 60 (TWEEN™60), polysorbate 65 (TWEEN™65),polysorbate 80 (TWEEN™80), polysorbate 85 (TWEEN™85), TRITON™ N-101,TRITON™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine,triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate(PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL),soy lecithin and a poloxamer. In one particular embodiment, thesurfactant is polysorbate 80. In some said embodiment, the finalconcentration of polysorbate 80 in the formulation is at least 0.0001%to 10% polysorbate 80 weight to weight (w/w). In some said embodiments,the final concentration of polysorbate 80 in the formulation is at least0.001% to 1% polysorbate 80 weight to weight (w/w). In some saidembodiments, the final concentration of polysorbate 80 in theformulation is at least 0.01% to 1% polysorbate 80 weight to weight(w/w). In other embodiments, the final concentration of polysorbate 80in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09% or 0.1% polysorbate 80 (w/w). In another embodiment, thefinal concentration of the polysorbate 80 in the formulation is 1%polysorbate 80 (w/w).

In certain embodiments, the immunogenic composition of the invention hasa pH of 5.5 to 7.5, more preferably a pH of 5.6 to 7.0, even morepreferably a pH of 5.8 to 6.0.

In one embodiment, the present invention provides a container filledwith any of the immunogenic compositions disclosed herein. In oneembodiment, the container is selected from the group consisting of avial, a syringe, a flask, a fermentor, a bioreactor, a bag, a jar, anampoule, a cartridge and a disposable pen. In certain embodiments, thecontainer is siliconized.

In an embodiment, the container of the present invention is made ofglass, metals (e.g., steel, stainless steel, aluminum, etc.) and/orpolymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers).In an embodiment, the container of the present invention is made ofglass.

In one embodiment, the present invention provides a syringe filled withany of the immunogenic compositions disclosed herein. In certainembodiments, the syringe is siliconized and/or is made of glass.

A typical dose of the immunogenic composition of the invention forinjection has a volume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1mL, even more preferably a volume of about 0.5 mL.

Therefore the container or syringe as defined above is filed with avolume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL, even morepreferably a volume of about 0.5 mL of any of the immunogeniccompositions defined herein.

6 Uses of the Immunogenic Compositions of the Invention

In an embodiment, the immunogenic compositions disclosed herein are foruse as a medicament.

The immunogenic compositions described herein may be used in varioustherapeutic or prophylactic methods for preventing, treating orameliorating a bacterial infection, disease or condition in a subject.In particular, immunogenic compositions described herein may be used toprevent, treat or ameliorate a S. pneumoniae infection, disease orcondition in a subject.

Thus in one aspect, the invention provides a method of preventing,treating or ameliorating an infection, disease or condition associatedwith S. pneumoniae in a subject, comprising administering to the subjectan immunologically effective amount of an immunogenic composition of theinvention.

In some such embodiments, the infection, disease or condition isselected from the group consisting of pneumonia, sinusitis, otitismedia, acute otitis media, meningitis, bacteremia, sepsis, pleuralempyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis,peritonitis, pericarditis, mastoiditis, cellulitis, soft tissueinfection and brain abscess.

In an embodiment, the invention provides a method of inducing an immuneresponse to S. pneumoniae in a subject comprising administering to thesubject an immunologically effective amount of an immunogeniccomposition of the invention

In an embodiment, the immunogenic compositions disclosed herein are foruse as a vaccine. In such embodiments the immunogenic compositionsdescribed herein may be used to prevent a S. pneumoniae infection in asubject. Thus in one aspect, the invention provides a method ofpreventing an infection by S. pneumoniae in a subject comprisingadministering to the subject an immunologically effective amount of animmunogenic composition of the invention. In some such embodiments, theinfection is selected from the group consisting of pneumonia, sinusitis,otitis media, acute otitis media, meningitis, bacteremia, sepsis,pleural empyema, conjunctivitis, osteomyelitis, septic arthritis,endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, softtissue infection and brain abscess. In one aspect, the subject to bevaccinated is a mammal, such as a human, cat, sheep, pig, horse, bovineor dog.

In one aspect, the immunogenic compositions disclosed herein are for usein a method of preventing, treating or ameliorating an infection,disease or condition associated with S. pneumoniae in a subject. In somesuch embodiments, the infection, disease or condition is selected fromthe group consisting of pneumonia, sinusitis, otitis media, acute otitismedia, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis,osteomyelitis, septic arthritis, endocarditis, peritonitis,pericarditis, mastoiditis, cellulitis, soft tissue infection and brainabscess.

In an embodiment, the immunogenic compositions disclosed herein are foruse as a vaccine. In such embodiments the immunogenic compositionsdescribed herein may be used to prevent a S. pneumoniae infection in asubject. Thus in one aspect, the immunogenic compositions disclosedherein are for use in a method of preventing, an infection by S.pneumoniae in a subject. In some such embodiments, the infection isselected from the group consisting of pneumonia, sinusitis, otitismedia, acute otitis media, meningitis, bacteremia, sepsis, pleuralempyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis,peritonitis, pericarditis, mastoiditis, cellulitis, soft tissueinfection and brain abscess. In one aspect, the subject to be vaccinatedis a mammal, such as a human, cat, sheep, pig, horse, bovine or dog.

The immunogenic compositions of the present invention can be used toprotect or treat a human susceptible to pneumococcal infection, by meansof administering the immunogenic compositions via a systemic or mucosalroute. In an embodiment, the immunogenic compositions disclosed hereinare administered by intramuscular, intraperitoneal, intradermal orsubcutaneous routes. In an embodiment, the immunogenic compositionsdisclosed herein are administered by intramuscular, intraperitoneal,intradermal or subcutaneous injection. In an embodiment, the immunogeniccompositions disclosed herein are administered by intramuscular orsubcutaneous injection.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when administeredto a subject, are able to induce the formation of antibodies capable ofbinding to S. pneumonia serotype 15B, 15A and/or 15C as measured by astandard ELISA assay. In an embodiment, the immunogenic composition ofthe present disclosure comprising at least one glycoconjugate from S.pneumoniae serotype 15B (such as the glycoconjugates of section 1.3.4above), when administered to a subject, are able to induce the formationof antibodies capable of binding to S. pneumonia serotype 15B, and 15Cas measured by a standard ELISA assay.

In the ELISA (Enzyme-linked Immunosorbent Assay) method, antibodies fromthe sera of vaccinated subjects are incubated with polysaccharides whichhave been adsorbed to a solid support. The bound antibodies are detectedusing enzyme-conjugated secondary detection antibodies.

In an embodiment said standard ELISA assay is the standardized (WHO)ELISA assay as defined by the WHO in the ‘Training manual for Enzymelinked immunosorbent assay for the quantitation of Streptococcuspneumoniae serotype specific IgG (Pn PS ELISA).’ (accessible athttp://www.vaccine.uab.edu/ELISA%20protocol.pdf; accessed on Mar. 31,2014).

The ELISA measures type specific IgG anti-S. pneumoniae capsularpolysaccharide (PS) antibodies present in human serum. When dilutions ofhuman sera are added to type-specific capsular PS-coated microtiterplates, antibodies specific for that capsular PS bind to the microtiterplates. The antibodies bound to the plates are detected using a goatanti-human IgG alkaline phosphatase-labeled antibody followed by ap-nitrophenyl phosphate substrate. The optical density of the coloredend product is proportional to the amount of anticapsular PS antibodypresent in the serum.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicitIgG antibodies in human which are capable of binding S. pneumoniaeserotype 15B polysaccharide at a concentration of at least 0.05, 0.1,0.2, 0.3, 0.35, 0.4 or 0.5 μg/ml as determined by ELISA assay.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicitIgG antibodies in human which are capable of binding S. pneumoniaeserotype 15C polysaccharide at a concentration of at least 0.05, 0.1,0.2, 0.3, 0.35, 0.4 or 0.5 μg/ml as determined by ELISA assay.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicitIgG antibodies in human which are capable of binding S. pneumoniaeserotypes 15B and 15C polysaccharide at a concentration of at least0.05, 0.1, 0.2, 0.3, 0.35, 0.4 or 0.5 μg/ml as determined by ELISAassay.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when administeredto a subject, are able to induce the formation of antibodies capable ofkilling S. pneumonia serotype 15B in an opsonophagocytosis assay asdisclosed herein (such as the OPA assay of Example 12).

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when tested in anOPA assay as disclosed herein (such as the OPA assay of Example 12), hasan OPA titer greater than the OPA titer obtained with an unconjugatednative S. pneumonia serotype 15B capsular polysaccharide.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when administeredto a subject, are able to induce the formation of antibodies capable ofkilling S. pneumonia serotype 15C in an opsonophagocytosis assay asdisclosed herein (such as the OPA assay of Example 12). In anembodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when tested in anOPA assay as disclosed herein (such as the OPA assay of Example 12), hasan OPA titer greater than the OPA titer obtained with an unconjugatednative S. pneumonia serotype 15B capsular polysaccharide.

The pneumococcal opsonophagocytic assay (OPA), which measures killing ofS. pneumoniae cells by phagocytic effector cells in the presence offunctional antibody and complement, is considered to be an importantsurrogate for evaluating the effectiveness of pneumococcal vaccines.

Opsonophagocytic assay (OPA) can be conducted by incubating together amixture of Streptococcus pneumoniae cells, a heat inactivated humanserum to be tested, differentiated HL-60 cells (phagocytes) and anexogenous complement source (e.g. baby rabbit complement).Opsonophagocytosis proceeds during incubation and bacterial cells thatare coated with antibody and complement are killed uponopsonophagocytosis. Colony forming units (cfu) of surviving bacteriathat escape from opsonophagocytosis are determined by plating the assaymixture. The OPA titer is defined as the reciprocal dilution thatresults in a 50% reduction in bacterial count over control wells withouttest serum. The OPA titer is interpolated from the two dilutions thatencompass this 50% killing cut-off.

An endpoint titer of 1:8 or greater is considered a positive result inthese killing type OPA.

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), is able to elicita titer of at least 1:8 against S. pneumoniae serotype 15B in at least50% of the subjects as determined by opsonophagocytic killing assay(OPA). In an embodiment, the immunogenic composition of the presentdisclosure comprising at least one glycoconjugate from S. pneumoniaeserotype 15B (such as the glycoconjugates of section 1.3.4 above) isable to elicit a titer of at least 1:8 against S. pneumoniae serotype15B in at least 60%, 70%, 80%, 90%, or at least 93% of the subjects asdetermined by opsonophagocytic killing assay (OPA).

In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicit atiter of at least 1:8 against S. pneumoniae serotype 15C in at least 50%of the subjects as determined by opsonophagocytic killing assay (OPA).In an embodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above) is able to elicit atiter of at least 1:8 against S. pneumoniae serotype 15C in at least60%, 70%, 80%, 90%, or at least 95% of the subjects as determined byopsonophagocytic killing assay (OPA).

In a further aspect, the present disclosure provides a method oftreating or preventing a S. pneumoniae infection, disease or conditionassociated with S. pneumoniae serotype 15A, 15B and/or 15C in a subject,the method comprising the step of administering a therapeutically orprophylactically effective amount of any of the immunogenic compositionsof the present disclosure comprising at least one glycoconjugate from S.pneumoniae serotype 15B (such as the glycoconjugates of section 1.3.4above). In an embodiment, the immunogenic composition of the presentdisclosure comprising at least one glycoconjugate from S. pneumoniaeserotype 15B (such as the glycoconjugates of section 1.3.4 above), whenadministered to a subject, induces the formation of antibodies capableof binding to S. pneumoniae serotype 15B, 15A and/or 15C. In anembodiment, the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above), when administeredto a subject, induces the formation of antibodies capable of killing S.pneumoniae serotype 15B, 15C and/or 15A in an opsonophagocytosis assayas disclosed herein (such as the OPA assay of Example 12).

One embodiment of the disclosure provides a method of protecting asubject against an infection with S. pneumoniae serotype 15C, or amethod of preventing infection with S. pneumoniae serotype 15C, or amethod of reducing the severity of or delaying the onset of at least onesymptom associated with an infection caused by S. pneumoniae serotype15C, the methods comprising administering to a subject an immunogenicamount of any of the immunogenic composition of the present disclosurecomprising at least one glycoconjugate from S. pneumoniae serotype 15B(such as the glycoconjugates of section 1.3.4 above). One embodiment ofthe disclosure provides a method of treating or preventing a S.pneumoniae infection, disease or condition associated with S. pneumoniaeserotype 15A, 15B and/or 15C (preferably 15B and/or 15C, more preferably15B) in a subject, the method comprising the step of administering atherapeutically or prophylactically effective amount of any of theimmunogenic composition of the present disclosure comprising at leastone glycoconjugate from S. pneumoniae serotype 15B (such as theglycoconjugates of section 1.3.4 above) to the subject. Anotherembodiment provides a method of treating or preventing a S. pneumoniaeinfection, disease or condition associated with a S. pneumoniae serotype15A, 15B and/or 15C (preferably 15B and/or 15C, more preferably 15B) ina subject, the method comprising generating a polyclonal or monoclonalantibody preparation from any of the immunogenic composition of thepresent disclosure comprising at least one glycoconjugate from S.pneumoniae serotype 15B (such as the glycoconjugates of section 1.3.4above), and using said antibody preparation to confer passive immunityto the subject.

In one embodiment, the disclosure relates to the use of any of theimmunogenic composition of the present disclosure comprising at leastone glycoconjugate from S. pneumoniae serotype 15B (such as theglycoconjugates of section 1.3.4 above) for the manufacture of amedicament for protecting a subject against an infection with S.pneumoniae, and/or preventing infection with S. pneumoniae, and/orreducing the severity of or delaying the onset of at least one symptomassociated with an infection caused by S. pneumoniae, and/or protectinga subject against an infection with S. pneumoniae serotype 15A, 15Band/or 15C (preferably 15B and/or 15C, more preferably 15B) and/orpreventing infection with S. pneumoniae serotype 15A, 15B and/or 15C(preferably 15B and/or 15C, more preferably 15B), and/or reducing theseverity of or delaying the onset of at least one symptom associatedwith an infection caused by S. pneumoniae serotype 15A, 15B and/or 15C(preferably 15B and/or 15C, more preferably 15B).

In one embodiment, the disclosure relates to the use of any of theimmunogenic composition of the present disclosure comprising at leastone glycoconjugate from S. pneumoniae serotype 15B (such as theglycoconjugates of section 1.3.4 above) for protecting a subject againstan infection with S. pneumoniae, and/or preventing infection with S.pneumoniae, and/or reducing the severity of or delaying the onset of atleast one symptom associated with an infection caused by S. pneumoniae,and/or protecting a subject against an infection with S. pneumoniaeserotype 15A, 15B and/or 15C (preferably 15B and/or 15C, more preferably15B) and/or preventing infection with S. pneumoniae serotype 15A, 15Band/or 15C (preferably 15B and/or 15C, more preferably 15B), and/orreducing the severity of or delaying the onset of at least one symptomassociated with an infection caused by S. pneumoniae serotype 15A, 15Band/or 15C (preferably 15B and/or 15C, more preferably 15B).

7 Subject to be Treated with the Immunogenic Compositions of theInvention

As disclosed herein, the immunogenic compositions described herein maybe used in various therapeutic or prophylactic methods for preventing,treating or ameliorating a bacterial infection, disease or condition ina subject.

In a preferred embodiment, said subject is a human. In a most preferredembodiment, said subject is a newborn (i.e., under three months of age),an infant (i.e., from 3 months to one year of age) or a toddler (i.e.,from one year to four years of age).

In an embodiment, the immunogenic compositions disclosed herein are foruse as a vaccine.

In such embodiment, the subject to be vaccinated may be less than 1 yearof age. For example, the subject to be vaccinated can be about 1, about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11 or about 12 months of age. In an embodiment, the subject tobe vaccinated is about 2, about 4 or about 6 months of age. In anotherembodiment, the subject to be vaccinated is less than 2 years of age.For example the subject to be vaccinated can be about 12 to about 15months of age. In some cases, as little as one dose of the immunogeniccomposition according to the invention is needed, but under somecircumstances, a second, third or fourth dose may be given (see section8 below).

In an embodiment of the present invention, the subject to be vaccinatedis a human adult 50 years of age or older, more preferably a human adult55 years of age or older. In an embodiment, the subject to be vaccinatedis a human adult 65 years of age or older, 70 years of age or older, 75years of age or older or 80 years of age or older.

In an embodiment the subject to be vaccinated is an immunocompromisedindividual, in particular a human. An immunocompromised individual isgenerally defined as a person who exhibits an attenuated or reducedability to mount a normal humoral or cellular defense to challenge byinfectious agents.

In an embodiment of the present invention, the immunocompromised subjectto be vaccinated suffers from a disease or condition that impairs theimmune system and results in an antibody response that is insufficientto protect against or treat pneumococcal disease.

In an embodiment, said disease is a primary immunodeficiency disorder.Preferably, said primary immunodeficiency disorder is selected from thegroup consisting of: combined T- and B-cell immunodeficiencies, antibodydeficiencies, well-defined syndromes, immune dysregulation diseases,phagocyte disorders, innate immunity deficiencies, autoinflammatorydisorders, and complement deficiencies. In an embodiment, said primaryimmunodeficiency disorder is selected from the one disclosed on page 24,line 11, to page 25, line 19, of WO 2010/125480.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated suffers from a diseaseselected from the groups consisting of: HIV-infection, acquiredimmunodeficiency syndrome (AIDS), cancer, chronic heart or lungdisorders, congestive heart failure, diabetes mellitus, chronic liverdisease, alcoholism, cirrhosis, spinal fluid leaks, cardiomyopathy,chronic bronchitis, emphysema, chronic obstructive pulmonary disease(COPD), spleen dysfunction (such as sickle cell disease), lack of spleenfunction (asplenia), blood malignancy, leukemia, multiple myeloma,Hodgkin's disease, lymphoma, kidney failure, nephrotic syndrome andasthma.

In an embodiment of the present invention, the immunocompromised subjectto be vaccinated suffers from malnutrition.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated is taking a drug or treatmentthat lowers the body's resistance to infection. In an embodiment, saiddrug is selected from the one disclosed on page 26, line 33, to page 26,line 4, of WO 2010/125480.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated is a smoker.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated has a white blood cell count(leukocyte count) below 5×10⁹ cells per liter, or below 4×10⁹ cells perliter, or below 3×10⁹ cells per liter, or below 2×10⁹ cells per liter,or below 1×10⁹ cells per liter, or below 0.5×10⁹ cells per liter, orbelow 0.3×10⁹ cells per liter, or below 0.1×10⁹ cells per liter.

White blood cell count (leukocyte count): The number of white bloodcells (WBC) in the blood. The WBC is usually measured as part of the CBC(complete blood count). White blood cells are the infection-fightingcells in the blood and are distinct from the red (oxygen-carrying) bloodcells known as erythrocytes. There are different types of white bloodcells, including neutrophils (polymorphonuclear leukocytes; PMN), bandcells (slightly immature neutrophils), T-type lymphocytes (T-cells),B-type lymphocytes (B-cells), monocytes, eosinophils, and basophils. Allthe types of white blood cells are reflected in the white blood cellcount. The normal range for the white blood cell count is usuallybetween 4,300 and 10,800 cells per cubic millimeter of blood. This canalso be referred to as the leukocyte count and can be expressed ininternational units as 4.3-10.8×10⁹ cells per liter.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated suffers from neutropenia. Ina particular embodiment of the present invention, the immunocompromisedsubject to be vaccinated has a neutrophil count below 2×10⁹ cells perliter, or below 1×10⁹ cells per liter, or below 0.5×10⁹ cells per liter,or below 0.1×10⁹ cells per liter, or below 0.05×10⁹ cells per liter.

A low white blood cell count or “neutropenia” is a conditioncharacterized by abnormally low levels of neutrophils in the circulatingblood. Neutrophils are a specific kind of white blood cell that help toprevent and fight infections. The most common reason that cancerpatients experience neutropenia is as a side effect of chemotherapy.Chemotherapy-induced neutropenia increases a patient's risk of infectionand disrupts cancer treatment.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated has a CD4+ cell count below500/mm³, or CD4+ cell count below 300/mm³, or CD4+ cell count below200/mm³, CD4+ cell count below 100/mm³, CD4+ cell count below 75/mm³, orCD4+ cell count below 50/mm³.

CD4 cell tests are normally reported as the number of cells in mm³.Normal CD4 counts are between 500 and 1,600, and CD8 counts are between375 and 1,100. CD4 counts drop dramatically in people with HIV.

In an embodiment of the invention, any of the immunocompromised subjectsdisclosed herein is a human male or a human female.

8 Regimen

In some cases, as little as one dose of the immunogenic compositionaccording to the invention is needed, but under some circumstances, suchas conditions of greater immune deficiency, a second, third or fourthdose may be given. Following an initial vaccination, subjects canreceive one or several booster immunizations adequately spaced.

In an embodiment, the schedule of vaccination of the immunogeniccomposition according to the invention is a single dose. In a particularembodiment, said single dose schedule is for healthy persons being atleast 2 years of age.

In an embodiment, the schedule of vaccination of the immunogeniccomposition according to the invention is a multiple dose schedule. In aparticular embodiment, said multiple dose schedule consists of a seriesof 2 doses separated by an interval of about 1 month to about 2 months.In a particular embodiment, said multiple dose schedule consists of aseries of 2 doses separated by an interval of about 1 month, or a seriesof 2 doses separated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses separated by an interval of about 1 month to about 2 months.In another embodiment, said multiple dose schedule consists of a seriesof 3 doses separated by an interval of about 1 month, or a series of 3doses separated by an interval of about 2 months.

In another embodiment, said multiple dose schedule consists of a seriesof 3 doses separated by an interval of about 1 month to about 2 monthsfollowed by a fourth dose about 10 months to about 13 months after thefirst dose. In another embodiment, said multiple dose schedule consistsof a series of 3 doses separated by an interval of about 1 monthfollowed by a fourth dose about 10 months to about 13 months after thefirst dose, or a series of 3 doses separated by an interval of about 2months followed by a fourth dose about 10 months to about 13 monthsafter the first dose.

In an embodiment, the multiple dose schedule consists of at least onedose (e.g., 1, 2 or 3 doses) in the first year of age followed by atleast one toddler dose.

In an embodiment, the multiple dose schedule consists of a series of 2or 3 doses separated by an interval of about 1 month to about 2 months(for example 28-56 days between doses), starting at 2 months of age, andfollowed by a toddler dose at 12-18 months of age. In an embodiment,said multiple dose schedule consists of a series of 3 doses separated byan interval of about 1 month to about 2 months (for example 28-56 daysbetween doses), starting at 2 months of age, and followed by a toddlerdose at 12-15 months of age. In another embodiment, said multiple doseschedule consists of a series of 2 doses separated by an interval ofabout 2 months, starting at 2 months of age, and followed by a toddlerdose at 12-18 months of age.

In an embodiment, the multiple dose schedule consists of a 4-dose seriesof vaccine at 2, 4, 6, and 12-15 months of age.

In an embodiment, a prime dose is given at day 0 and one or more boostsare given at intervals that range from about 2 to about 24 weeks,preferably with a dosing interval of 4-8 weeks.

In an embodiment, a prime dose is given at day 0 and a boost is givenabout 3 months later.

As used herein, the term “about” means within a statistically meaningfulrange of a value, such as a stated concentration range, time frame,molecular weight, temperature or pH. Such a range can be within an orderof magnitude, typically within 20%, more typically within 10%, and evenmore typically within 5% or within 1% of a given value or range.Sometimes, such a range can be within the experimental error typical ofstandard methods used for the measurement and/or determination of agiven value or range. The allowable variation encompassed by the term“about” will depend upon the particular system under study, and can bereadily appreciated by one of ordinary skill in the art. Whenever arange is recited within this application, every whole number integerwithin the range is also contemplated as an embodiment of thedisclosure.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting essentially of”, “consist essentially of”, “consistsessentially of”, “consisting of”, “consist of” and “consists of”,respectively, in every instance.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

The invention is illustrated in the accompanying examples. The examplesbelow are carried out using standard techniques, which are well knownand routine to those of skill in the art, except where otherwisedescribed in detail. The examples are illustrative, but do not limit theinvention.

EXAMPLE Example 1 General Process for Preparation of eTEC LinkedGlycoconjugates

Activation of Saccharide and Thiolation with Cystamine Dihydrochloride

The saccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO).Moisture content of the solution is determined by Karl Fischer (KF)analysis and adjusted to reach a moisture content of between 0.1% and0.4%, typically 0.2%.

To initiate the activation, a solution of1,1′-carbonyl-di-1,2,4-triazole (CDT) or 1,1′-carbonyldiimidazole (CDI)is freshly prepared at a concentration of 100 mg/mL in DMSO. Thesaccharide is activated with various amounts of CDT/CDI (1-10 molarequivalents) and the reaction is allowed to proceed for 1 hour at 23±2°C. The activation level may be determined by HPLC. Cystaminedihydrochloride is freshly prepared in anhydrous DMSO at a concentrationof 50 mg/mL. The activated saccharide is reacted with 1 molarequivalents (mol. eq.) of cystamine dihydrochloride. Alternatively, theactivated saccharide is reacted with 1 mol. eq. of cysteaminehydrochloride. The thiolation reaction is allowed to proceed for 21±2hours at 23±2° C., to produce a thiolated saccharide. The thiolationlevel is determined by the added amount of CDT/CDI.

Residual CDT/CDI in the activation reaction solution is quenched by theaddition of 100 mM sodium tetraborate, pH 9.0 solution. Calculations areperformed to determine the added amount of tetraborate and to adjust thefinal moisture content to be up to 1-2% of total aqueous.

Reduction and Purification of Activated Thiolated Saccharide

The thiolated saccharide reaction mixture is diluted 10-fold by additionto pre-chilled 5 mM sodium succinate in 0.9% saline, pH 6.0 and filteredthrough a 5 μm filter. Dialfiltration of thiolated saccharide isperformed against 40-fold diavolume of WFI. To the retentate a solutionof tris(2-carboxyethyl)phosphine (TCEP), 1-5 mol. eq., is added afterdilution by 10% volume of 0.1M sodium phosphate buffer, pH 6.0. Thisreduction reaction is allowed to proceed for 20±2 hours at 5±3° C.Purification of the activated thiolated saccharide is performedpreferably by ultrafiltration/dialfiltration of against pre-chilled 10mM sodium phosphate monobasic, pH 4.3. Alternatively, the thiolatedsaccharide is purified by standard size exclusion chromatographic (SEC)procedures or ion exchange chromatographic methods. An aliquot ofactivated thiolated saccharide retentate is pulled to determine thesaccharide concentration and thiol content (Ellman) assays.

Alternative Reduction and Purification of Activated Thiolated Saccharide

As an alternative to the purification procedure described above,activated thiolated saccharide was also purified as below.

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 5-10 mol. eq., was added andallowed to proceed for 3±1 hours at 23±2° C. The reaction mixture wasthen diluted 5-fold by addition to pre-chilled 5 mM sodium succinate in0.9% saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltrationof thiolated saccharide was performed using 40-fold diavolume ofpre-chilled 10 mM sodium phosphate monobasic, pH 4.3. An aliquot ofactivated thiolated saccharide retentate was pulled to determine thesaccharide concentration and thiol content (Ellman) assays.

Activation and Purification of Bromoacetylated Carrier Protein

Free amino groups of the carrier protein are bromoacteylated by reactionwith a bromoacetylating agent, such as bromoacetic acidN-hydroxysuccinimide ester (BAANS), bromoacetylbromide, or anothersuitable reagent.

The carrier protein (in 0.1 M Sodium Phosphate, pH 8.0±0.2) is firstkept at 8±3° C., at about pH 7 prior to activation. To the proteinsolution, the N-hydroxysuccinimide ester of bromoacetic acid (BAANS) asa stock dimethylsulfoxide (DMSO) solution (20 mg/mL) is added in a ratioof 0.25-0.5 BAANS: protein (w/w). The reaction is gently mixed at 5±3°C. for 30-60 minutes. The resulting bromoacetylated (activated) proteinis purified, e.g., by ultrafiltration/diafiltration using 10 kDa MWCOmembrane using 10 mM phosphate (pH 7.0) buffer. Following purification,the protein concentration of the bromoacetylated carrier protein isestimated by Lowry protein assay.

The extent of activation is determined by total bromide assay byion-exchange liquid chromatography coupled with suppressed conductivitydetection (ion chromatography). The bound bromide on the activatedbromoacetylated protein is cleaved from the protein in the assay samplepreparation and quantitated along with any free bromide that may bepresent. Any remaining covalently bound bromine on the protein isreleased by conversion to ionic bromide by heating the sample inalkaline 2-mercaptoethanol.

Activation and Purification of Bromoacetylated CRM₁₉₇

CRM₁₉₇ was diluted to 5 mg/mL with 10 mM phosphate buffered 0.9% NaCl pH7 (PBS) and then made 0.1 M NaHCO₃, pH 7.0, using 1 M stock solution.BAANS was added at a CRM₁₉₇: BAANS ratio 1:0.35 (w:w) using a BAANSstock solution of 20 mg/mL DMSO. The reaction mixture was incubated atbetween 3° C. and 11° C. for 30 mins-1 hour then purified byultrafiltration/diafiltration using a 10K MWCO membrane and 10 mM SodiumPhosphate/0.9% NaCl, pH 7.0. The purified activated CRM₁₉₇ was assayedby the Lowry assay to determine the protein concentration and thendiluted with PBS to 5 mg/mL. Sucrose was added to 5% wt/vol as acryoprotectant and the activated protein was frozen and stored at −25°C. until needed for conjugation.

Bromoacetylation of lysine residues of CRM₁₉₇ was very consistent,resulting in the activation of 15 to 25 lysines from 39 lysinesavailable. The reaction produced high yields of activated protein.

Conjugation of Activated Thiolated Saccharide to Bromoacetylated CarrierProtein

Before starting the conjugation reaction, the reaction vessels arepre-cooled to 5° C. Bromoacetylated carrier protein and activatedthiolated saccharide are subsequently added and mixed at an agitationspeed of 150-200 rpm. The saccharide/protein input ratio is 0.9±0.1. Thereaction pH is adjusted to 8.0±0.1 with 1 M NaOH solution. Theconjugation reaction is allowed to proceed at 5° C. for 20±2 hours.

Capping of Residual Reactive Functional Groups

The unreacted bromoacetylated residues on the carrier protein arequenched by reacting with 2 mol. eq. of N-acetyl-L-cysteine as a cappingreagent for 3 hours at 5° C.

Residual free sulfhydryl groups are capped with 4 mol. eq. ofiodoacetamide (IAA) for 20 hours at 5° C.

Purification of eTEC-Linked Glycoconjudate

The conjugation reaction (post-IAA-capped) mixture is filtered through0.45 μm filter. Ultrafiltration/dialfiltration of the glycoconjugate isperformed against 5 mM succinate-0.9% saline, pH 6.0. The glycoconjugateretentate is then filtered through 0.2 μm filter. An aliquot ofglycoconjugate is pulled for assays. The remaining glycoconjugate isstored at 5° C.

Example 2 Preparation of Pn-33F eTEC Conjugates

Activation Process

Activation of Pn33F Polysaccharide

Pn-33F polysaccharide was compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture was shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized 33F polysaccharide was reconstituted inanhydrous dimethylsulfoxide (DMSO). Moisture content of the lyophilized33F/DMSO solution was determined by Karl Fischer (KF) analysis. Themoisture content was adjusted by adding WFI to the 33F/DMSO solution toreach a moisture content of 0.2%.

To initiate the activation, 1,1′-carbonyl-di-1,2,4-triazole (CDT) wasfreshly prepared as 100 mg/mL in DMSO solution. Pn33F polysaccharide wasactivated with various amounts of CDT prior to the thiolation step. TheCDT activation was carried out at 23±2° C. for 1 hour. The activationlevel was determined by HPLC (A220/A205). Sodium tetraborate, 100 mM, pH9.0 solution was added to quench any residual CDT in the activationreaction solution. Calculations are performed to determine the addedamount of tetraborate and to allow the final moisture content to be 1.2%of total aqueous. The reaction was allowed to proceed for 1 hour at23±2° C.

Thiolation of Activated Pn-33F Polysaccharide

Cystamine-dihydrochloride was freshly prepared in anhydrous DMSO and 1mol. eq. of cystamine dihydrochloride was added to the activatedpolysaccharide reaction solution. The reaction was allowed to proceedfor 21±3 hours at 23±2° C. The thiolated saccharide solution was diluted10-fold by addition to pre-chilled 5 mM sodium succinate in 0.9% saline,pH 6.0. The diluted reaction solution was filtered through a 5 μmfilter. Dialfiltration of thiolated Pn-33F polysaccharide was carriedout with 100K MWCO ultrafilter membrane cassettes, using Water forInjection (WFI).

Reduction and Purification of Activated Thiolated Pn-33F Polysaccharide

To the retentate a solution of tris(2-carboxyethyl)phosphine (TCEP), 5mol. eq., was added after dilution by 10% volume of 0.1 M sodiumphosphate buffer, pH 6.0. This reduction reaction was allowed to proceedfor 2±1 hours at 23±2° C. Dialfiltration of thiolated 33F polysaccharidewas carried out with 100K MWCO ultrafilter membrane cassettes.Diafiltration was performed against pre-chilled 10 mM sodium phosphate,pH 4.3. The thiolated 33F polysaccharide retentate was pulled for bothsaccharide concentration and thiol (Ellman) assays.

Alternative Reduction and Purification of Activated Thiolated Pn-33FPolysaccharide

As an alternative to the purification procedure described above, 33Factivated thiolated saccharide was also purified as follows.

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 5 mol. eq., was added and allowedto proceed for 3±1 hours at 23±2° C. The reaction mixture was thendiluted 5-fold by addition to pre-chilled 5 mM sodium succinate in 0.9%saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltration ofthiolated saccharide was performed using 40-fold diavolume ofpre-chilled 10 mM sodium phosphate monobasic, pH 4.3 with 100K MWCOultrafilter membrane cassettes. The thiolated 33F polysaccharideretentate was pulled for both saccharide concentration and thiol(Ellman) assays. A flow diagram of the activation process is provided inFIG. 8(A).

Conjugation Process

Conjugation of Thiolated Pn33F Polysaccharide to Bromoacetylated CRM₁₉₇

The CRM₁₉₇ carrier protein was activated separately by bromoacetylation,as described in Example 1, and then reacted with the activated Pn-33Fpolysaccharide for the conjugation reaction. Before starting theconjugation reaction, the reaction vessel was pre-cooled to 5° C.Bromoacetylated CRM₁₉₇ and thiolated 33F polysaccharide were mixedtogether in a reaction vessel at an agitation speed of 150-200 rpm. Thesaccharide/protein input ratio was 0.9±0.1. The reaction pH was adjustedto 8.0-9.0. The conjugation reaction was allowed to proceed at 5° C. for20±2 hours.

Capping of Reactive Groups on Bromoacetylated CRM₁₉₇ and Thiolated Pn33FPolysaccharide

The unreacted bromoacetylated residues on CRM₁₉₇ proteins were capped byreacting with 2 mol. eq. of N-acetyl-L-cysteine for 3 hours at 5° C.,followed by capping any residual free sulfhydryl groups of the thiolated33F-polysaccharide with 4 mol. eq. of iodoacetamide (IAA) for 20 hoursat 5° C.

Purification of eTEC-Linked Pn-33F Glycoconjugate

The conjugation solution was filtered through a 0.45 μm or 5 μm filter.Dialfiltration of the 33F glycoconjugate was carried out with 300K MWCOultrafilter membrane cassettes. Diafiltration was performed against 5 mMsuccinate-0.9% saline, pH 6.0. The Pn-33F glycoconjugate 300K retentatewas then filtered through a 0.22 μm filter and stored at 5° C. A flowdiagram of the conjugation process is provided in FIG. 8(B).

Results

The reaction parameters and characterization data for several batches ofPn-33F eTEC glycoconjugates are shown in Table 1. The CDTactivation-thiolation with cystamine dihydrochloride generatedglycoconjugates having from 63% to 90% saccharide yields and <1% to 13%free saccharides.

TABLE 1 Experimental Parameters and Characterization Data of Pn33F eTECConjugates Conjugate Batch 33F-1A 33F-2B 33F-3C 33F-4D 33F-5E 33F-6F33F-7G Activation level (mol of 0.21 0.13 0.164 0.103 0.183 0.22 0.19thiol/mol of polysaccharide) Activation level 21 13 16.4 10.3 18.3 22 19(% Thiol) Saccharide/Protein 0.75 1.0 0.75 1.0 1.0 0.75 0.80 (Input)ratio Saccharide yield (%) 69% 63% 71% 63% 69% 82% 90%Saccharide/Protein 1.3 1.7 1.2 1.9 1.6 1.1 1.5 Ratio Free Saccharide12.9%   7.7%  4.4%  7.2%  7.3%  <4% <4% MW by SEC-MALLS 2627 2561 43512981 3227 3719 5527 (kDa) CMCA/CMC 14.4/0 13.4/0 6.8/1.9 2.7/0.6 5.9/0.68.2/0 11.4/0.6 % K_(d) (≤0.3) N/A 85% 88% 75% 68% 67% 76% Acetylationlevel (mol 0.89 1.16 0.99 0.85 0.81 0.85 1.01 of acetate/mol ofpolysaccharide) N/A = not available

OPA Titers of Pn-33F eTEC Glycoconjugates to CRM₁₉₇

Pn-33F OPA titers in mice were determined under standard conditions(similar to the OPA procedures described below for 10A and 22Fconjugates). OPA titers (GMT with 95% CI) at four and seven weeks areshown in Table 2, demonstrating that the serotype 33F Pn glycoconjugateelicited OPA titers in a murine immunogenicity model.

TABLE 2 Pn-33F OPA Titers (GMT with 95% CI) 33F Pn Conjugate 0.001 μg0.01 μg 0.1 μg week 4 4 (4, 5)  37 (17, 82)  414 (234, 734) week 7 8 (5,13) 131 (54, 314) 17567 (9469, 32593)

Example 3 Preparation of Additional Pn-33F eTEC Conjugates

Additional Pn-33F eTEC Conjugates were generated using the processdescribed in Example 2. The reaction parameters and characterizationdata for these additional batches of Pn-33F eTEC glycoconjugates areshown in Table 3.

TABLE 3 Experimental Parameters and Characterization Data of furtherPn33F eTEC Conjugates Conjugate Batch 33F- 33F- 33F- 33F- 33F- 33F- 33F-33F- 33F- 8H 9I 10J 11K 12L 13M 14N 15O 16P Activation level 0.22 0.110.11 0.13 0.14 0.13 0.06 0.13 0.11 (mol of thiol/mol of polysaccharide)Saccharide/Protein 0.75 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 (Input) ratioSaccharide yield 78% 88% 89% 67% 69% 86% 81% 91% 88% (%)Saccharide/Protein 1.0 2.2 2.1 1.4 1.4 1.4 2.2 1.9 1.9 Ratio FreeSaccharide <1% 6.8%  5.9%  2.3%  3.6%  LOQ 8.2%  3.6%  6.6%  MW by SEC-4729 3293 3295 2246 2498 5539 3070 6009 3789 MALLS (kDa) CMCA/ 6.6/14.2/ 15.4/ 5.5/1 5.4/ NA/ 1.7/ 4.1/ 2.2/ CMC LOQ 2.1 2.1 1.1 LOQ 1.22.2 1.2 % K_(d) (≤0.3) 69% N/A N/A N/A N/A 88% 87% 87% 85% Acetylationlevel 0.86 0.93 0.87 1.01 0.99 0.71 0.78 0.8 0.82 (mol of acetate/mol ofpolysaccharide) LOQ = limit of quantitation; N/A = not available.

As shown above and in Table 3, several Pn-33F conjugates were obtainedusing the eTEC conjugation above. The eTEC chemistry allowed preparationof conjugates with high yield, low % free saccharide and high degree ofconjugation (conjugated lysines). Additionally, it was possible topreserve more than 80% of acetyl functionality using the eTECconjugation process.

Example 4 Evaluation of Pn-33F eTEC Glycoconjugates Stability: % FreeSaccharide Trends

Aliquots of conjugate batch 33F-2B (see table 1) were dispensed intopolypropylene tubes and stored at 4° C., 25° C., and 37° C.,respectively and monitored for trends in % free saccharide. The data (%free saccharide) are shown in Table 4. As shown in this Table, therewere no significant changes in the % free saccharide.

TABLE 4 % Free Saccharide Stability for Pn-33F eTEC Glycoconjugate at 4°C., 25° C. and 37° C. Free Saccharide (%) Time Lot# 0 1 wk 3 wks 1 M 2 M3 M 6 M 4° C. 33F-2B 7.7 N/A 8.3 N/A 9.7 11.2 13 25° C. 7.7 N/A 10.8 N/A11.8 N/A N/A 37° C. 7.7 12.1 N/A 13.4 N/A N/A N/A wk = week; M = month;N/A = not available.

The accelerated stability of another conjugate lot (Batch 33F-3C) wasalso conducted at 37° C. up to 1 month. As shown in Table 5, there wasno significant change to % free saccharide at 37° C., up to 1 month.

TABLE 5 % Free Saccharide Stability for Pn-33F eTEC Glycoconjugate at37° C. Free Saccharide (%) Time 0 1 day 1 wk 2 wks 1 M Lot# 37° C.33F-3C 4.4 5.9 6.4 7.1 7.2

To further confirm the stability of eTEC conjugates, additionalconjugate batches (33F-3C and 33F-5E (see Table 1)) stored at 4° C. weremonitored up to approximately one year, for potential trends in % freesaccharide. As shown in Table 6, there were no significant changes in %free saccharide levels for the conjugates stored at 4° C. for anextended period up to approximately one year.

TABLE 6 % Free Saccharide Stability Results for Pn-33F eTECGlycoconjugates at 4° C. Free Saccharide (%) Time 0 3 M 4 M 12 M 14 MLot# 4° C. 33F-3C 4.4 N/A 5.3 N/A 7.6 33F-5E 7.3 6.3 N/A 7.4 N/A M =month; N/A = not available

The Serotype 33F conjugates generated by 33F eTEC chemistry weredemonstrated to be stable without noticeable degradation as monitored bythe free saccharide trends at various temperatures (real time andaccelerated).

Example 5 Preparation of Pn-8 Conjugates to CRM₁₉₇

Preparation of Pn-8 RAC/DMSO Glycoconjugates

Frozen polysaccharide was thawed and transferred to the reaction vessel.2 M acetic acid and WFI (Water for Injection) was added to thepolysaccharide solution to achieve a final polysaccharide concentrationof about 2.5 g/L and a final acetic acid concentration of 0.2 M.

Hydrolysis of the Polysaccharide

The native polysaccharide was chemically hydrolyzed prior to activation.The diluted polysaccharide solution was heated to 70° C., and then heldthis temperature for 3.5 hours.

Oxidation of the Polysaccharide

Oxidation of polysaccharide was initiated by the addition of sodiumperiodate solution and the reaction kept to proceed for 20 hrs at 23° C.

Purification of Activated Polysaccharide

The activated polysaccharide was concentrated using ultrafiltrationcassettes. Diafiltration was performed against 20-fold diavolume of WFI.

Lyophilization

The activated polysaccharide is compounded with sucrose to a ratio of 25grams of sucrose per gram of activated polysaccharide. The bottlescontaining the activated saccharide and sucrose are shell frozen inethanol baths and lyophilized.

Conjugation of Activated Polysaccharide to CRM₁₉₇ and Capping

Lyophilized activated polysaccharide was reconstituted to 2 mg/mL inDMSO. DMSO was added to lyophilized CRM₁₉₇ for reconstitution.Reconstituted CRM₁₉₇ was added to the reconstituted activatedpolysaccharide. Conjugation was then initiated by adding sodiumcyanoborohydride to the reaction mixture and was incubated at 23° C. for24 hrs. Termination of conjugation reaction is done by adding 2 MEq ofsodium borohydride. This capping reaction proceeded for 3 hrs at 23° C.

Purification of Conjugate

The conjugate solution was then diluted into chilled 5 mM succinate-0.9%saline (pH 6.0), filtered, concentrated to 2-4 g/L using 300K cellulosemembranes, and a first-stage diafiltration was performed against 5 mMsuccinate-0.9% saline (pH6.0). A final purification step was done bydiafiltration with 5 mM succinate-0.9% saline, pH 6.0 buffer. After thediafiltration is completed, the purified conjugate was transferred to acollection tank through a 0.22 μm filter.

Dilution of the Monovalent Bulk Conjugate

The conjugate was diluted further with 5 mM succinate/0.9% saline (pH6), to a target saccharide concentration of 0.5 mg/mL. Final 0.22 μmfiltration step was completed to prepare the monovalent bulk conjugate(MBC) product for formulation.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and Meq of sodium cyanoborohydride).Characterization for representative Pn-8 glycoconjugates to CRM₁₉₇ isprovided in Table 7.

TABLE 7 Characterization of Pn8-CRM₁₉₇ Conjugates Sample No. 1 2 3 4 5 67 8 9 Activated Saccharide 267 270 352 65 233 340 113 250 230 MW byMALLS (kDa) Saccaride/Protein 0.81 0.84 0.5 2.7 1.15 1.0 0.81 0.64 0.42Ratio MW by SEC-MALLS 12200 8670 3460 3379 4748 4255 5470 9924 6787(kDa)

The Opsonophagocytic activity (OPA) titers for Serotype 8-CRM₁₉₇conjugates in mice were determined in mice under standard conditions(similar to the OPA procedures described below for 10A and 22Fconjugates). OPA titers (geometric mean titer (GMT) with 95% confidenceinterval (CI)) at four weeks at different doses are shown in Table 8 and9 (two separate experiments), demonstrating that the serotype 8conjugate (Samples 1-9; also see Table 7 for characterization data ofthese conjugates) elicited OPA titers in a murine immunogenicity model.

As shown in Table 8, serotype 8 conjugates were shown to havesignificantly higher antibody titers, compared to the controlunconjugated polysaccharide which had poor antibody titers.

TABLE 8 Immunogenicity of Serotype 8-CRM₁₉₇ Conjugates OPA GMT (95% CI)Sample No. 0.001 μg 0.01 μg 0.1 μg 1 17 (10, 30)  88 (47, 165) 1344(896, 2016) 2  7 (4, 11) 184 (87, 387) 1934 (1313, 2847) 3  4 (4, 4)  17(9, 30)  779 (345, 1757) 4  5 (4, 7)  74 (41, 136)  558 (311, 1001)Unconjugated PS  13 (3, 55)

TABLE 9 Immunogenicity of Serotype 8-CRM₁₉₇ Conjugates Sample OPA GMT(95% CI) No. 0.001 μg 0.01 μg 5  8 (5, 12) 322 (208, 498) 6 12 (8, 19)264 (129, 537) 7 12 (7, 21) 521 (366, 743) 8 19 (10, 38) 404 (238, 687)9 33 (14, 80) 686 (380, 1237) 2 13 (7, 23) 177 (94, 336)

The overall data generated from conjugates prepared by the abovereductive amination process demonstrated that it allowed preparingconjugates with good conjugation yield, low % free saccharide and withgood stability. Additionally, the prepared conjugates elicited good OPAtiters in a murine immunogenicity model.

Example 6 Preparation of Serotype 10A Polysaccharide—CRM₁₉₇ Conjugate

Preparation of Isolated S. pneumoniae Serotype 10A Polysaccharide

Serotype 10A capsular polysaccharides can be obtained directly frombacteria using isolation procedures known to one of ordinary skill inthe art (see for example methods disclosed in U.S. Patent App. Pub. Nos.2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340,and 2008/0102498 and WO 2008/118752). Streptococcus pneumoniae serotype10A were grown in a seed bottle and then transferred to a seedfermentor. Once the targeted optical density was reached, the cells weretransferred to a production fermentor. The fermentation broth wasinactivated by the addition of N-lauroyl sarcosine and purified byultrafiltration and diafiltration.

Oxidation of Isolated Streptococcus pneumoniae Serotype 10A CapsularPolysaccharide

A calculated volume of 0.1 M potassium phosphate buffer (pH 6.0) andwater-for-injection (WFI) was added to the polysaccharide solution toachieve a final polysaccharide concentration of 2.5 g/L and a finalconcentration of 25 mM potassium phosphate buffer, if required pH wasadjusted to 6.0, approximately. The diluted polysaccharide was thencooled to 5° C. Oxidation was initiated by the addition of 0.25 molarequivalents (MEq) of sodium periodate solution. The oxidation reactiontime was approximately 4 hrs at 5° C. The oxidation reaction wasquenched with 1 MEq of 2,3-butanediol under continuous stirring at 5° C.for 1-2 hrs.

After reaching the target reaction time, the activated polysaccharidewas concentrated using 30K MWCO Millipore ultrafiltration cassettes. Thediafiltration was then performed against 20-fold diavolume of WFI. Thepurified activated polysaccharide was stored at 5° C. The purifiedactivated saccharide is characterized inter alia by (i) Molecular Weightby SEC-MALLS and (ii) Degree of Oxidation.

Conjugation of Activated S. pneumoniae Serotype 10A Polysaccharide withCRM₁₉₇

The conjugation process consisted of the following steps:

a. Compounding with sucrose excipient, and lyophilization;

b. Reconstitution of the lyophilized polysaccharide and CRM₁₉₇;

c. Conjugation of activated polysaccharide to CRM₁₉₇ and capping; and

d. Purification of the conjugate

a. Compounding with Sucrose

The activated polysaccharide is compounded with sucrose to a ratio of 25g of sucrose per gram of activated polysaccharide. The bottle ofcompounded mixture was then lyophilized. Following lyophilization,bottles containing lyophilized activated polysaccharide were stored at−20° C.

b. Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in anhydrousdimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide,the same amount of DMSO was added to the calculated CRM₁₉₇ forreconstitution.

c. Conjugation of Activated Polysaccharide to CRM₁₉₇ and Capping

Reconstituted CRM₁₉₇ (in DMSO) was added to the reconstituted activatedpolysaccharide in the conjugation reactor. The final polysaccharideconcentration is 1 g/L. Conjugation was performed by adding 1.2 MEq ofsodium cyanoborohydride to the reaction mixture. The reaction wasincubated and at 23° C. for 24 hrs. Termination of conjugation reactionis done by adding 2 MEq of sodium borohydride. The capping reaction wasincubated at 23° C. for 3 hrs.

Termination of conjugation reaction is done by adding 2 MEq of sodiumborohydride. This capping reaction proceeded for 3 hrs at 23° C.

d. Purification of Conjugate

The conjugate solution was then diluted into 5× (by volume) chilled 5 mMsuccinate-0.9% saline (pH 6.0) and a 20× diafiltration was performedusing 5 mM succinate-0.9% saline (pH6.0). After the initialdiafiltration was completed, the conjugate retentate was transferredthrough a 0.22 μm filter. The conjugate was diluted further with 5 mMsuccinate/0.9% saline (pH 6), and after the final 0.22 μm filtrationstep it was stored at 2-8° C.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and MEq of sodium cyanoborohydride). The abovechemistry allowed to generate serotype 10A conjugates which weredemonstrated to be stable without noticeable degradation as monitored bythe free saccharide trends at various temperatures (real time andaccelerated). Characterization for representative Pn-10A glycoconjugatesto CRM₁₉₇ is provided in Table 10.

TABLE 10 Characterization of Pn-10A-CRM₁₉₇ Conjugates Conjugate No. 1 23 4 5 6 DO 12.2 19.5 5.2 10.3 10.8 10.5 Activated 191 240 80 170 170 170Saccharide MW, kDa Input Ratio 1.0 1.0 1.0 1.1 1.1 1.1 % Yield 56 28.565 82 73 66 % Free 6.8 10.0 6.7 6.8 6.4 9.7 Saccharide Conjugate 38385810 4630 4034 3463 5540 MW, kDa Saccharide/ 0.82 0.88 0.85 1.1 1.2 1.0Protein Ratio Lys 7.4 3.7 13.1 6.9 6.7 6.1 modification AAA

The opsonophagocytic activity (OPA) titers for Serotype 10A-CRM₁₉₇conjugates in mice were determined under standard conditions. Groups ofthirty 6-7 week old female Swiss Webster mice were immunized with 0.001μg, 0.01 μg, or 0.1 μg of test conjugates via the subcutaneous route onweek 0. The mice were boosted with the same dose of conjugate on week 3and then bled at week 4. Serotype-specific OPAs were performed on week 4sera samples.

Opsonophagocytic activity (OPA) assays are used to measure functionalantibodies in murine sera specific for S. pneumonia serotype 10A. Testserum is set up in assay reactions that measure the ability of capsularpolysaccharide specific immunoglobulin to opsonize bacteria, triggercomplement deposition, thereby facilitating phagocytosis and killing ofbacteria by phagocytes. The OPA titer is defined as the reciprocaldilution that results in a 50% reduction in bacterial count over controlwells without test serum. The OPA titer is interpolated from the twodilutions that encompass this 50% killing cut-off.

OPA procedures were based on methods described in Hu et al. (2005) OlinDiagn Lab Immunol12 (2):287-295 with the following modifications. Testserum was serially diluted 2.5-fold and added to microtiter assayplates. Live serotype 10A target bacterial strains were added to thewells and the plates were shaken at 37° C. for 30 minutes.

Differentiated HL-60 cells (phagocytes) and baby rabbit serum (3- to4-week old, PEL-FREEZ®, 12.5% final concentration) were added to thewells, and the plates were shaken at 37° C. for 60 minutes. To terminatethe reaction, 80 μL of 0.9% NaCl was added to all wells, mixed, and a 10μL aliquot were transferred to the wells of MULTISCREEN® HTS HV filterplates (MILLIPORE®) containing 200 μL of water. Liquid was filteredthrough the plates under vacuum, and 150 μL of HYSOY® medium was addedto each well and filtered through. The filter plates were then incubatedat 37° C., 5% CO₂ overnight and were then fixed with Destain Solution(Bio-Rad Laboratories, Inc., Hercules, Calif.). The plates were thenstained with Coomassie Blue and destained once. Colonies were imaged andenumerated on a Cellular Technology Limited (CTL) (Shaker Heights, Ohio)IMMUNOSPOT® Analyzer. Raw colony counts were used to plot kill curvesand calculate OPA titers.

OPA titers (geometric mean titer (GMT) with 95% confidence interval(CI)) at four weeks at different doses are shown in Table 11,demonstrating that the serotype 10A conjugate (Samples 1-3; also seeTable 10 for characterization data of these conjugates) elicited OPAtiters in a murine immunogenicity model. As shown in Table 11, serotype10A conjugates were shown to have significantly higher OPA titers,compared to the control unconjugated polysaccharide, which had a poorOPA response.

TABLE 11 Immunogenicity of Serotype 10A-CRM₁₉₇ Conjugates OPA GMT (95%CI) Sample No. 0.001 μg 0.01 μg 0.1 μg 1  858 (556, 1324) 1015 (610,1691) 4461 (3065, 6494) 2 1411 (737, 2703)  796 (460, 1378) 2873 (1768,4842) 3  322 (180, 574) 1062 (528, 2135) 2618 (1415, 4842) Uncon-  602(193, 1882) jugated PS

Example 7 Conjugation of Pn Serotype-12F Using TEMPO/NCS

In order to improve the stability of serotype 12F-CRM₁₉₇glycoconjugates, alternate chemistries were explored using2,2,6,6-Tetramethyl-1-piperidinyloxy free radical (TEMPO) andN-Chlorosuccinimide (NCS) as the cooxidant to oxidize primary alcoholsto aldehyde groups. GC/MS analysis showed that the sites of oxidationwere different from that of periodate-mediated oxidation. In the case ofTEMPO-NCS oxidation, the α-D-Glcp and 2-Glcp were oxidized, whereasα-D-Galp was the major site of oxidation when periodate was used (seeFIG. 4). As described in further detail herein, TEMPO was used incatalytic amounts (≤0.1 molar equivalents) and the desired degree ofoxidation (DO) was achieved by varying the amounts of NCS used.Subsequently several conjugates were synthesized and characterized. Ingeneral, the production of Serotype 12F glycoconjugates was carried outin several phases, as follows:

a) Hydrolysis of Serotype 12F polysaccharide to molecular weights 50 kDato 500 kDa

b) Activation of Serotype 12F polysaccharide with TEMPO/NCS;

c) Purification of the activated polysaccharide;

d) Conjugation of activated Serotype 12F to CRM₁₉₇ protein; and

e) Purification of Serotype 12F-CRM₁₉₇ conjugates.

Hydrolysis and Oxidation of Serotype 12F

The hydrolysis of the polysaccharide was typically performed underacidic conditions with heating to obtain an average molecular weight inthe desired range of 100 kDa to 350 kDa. A typical experiment isdescribed below.

Hydrolysis

The Serotype 12F polysaccharide solution was added to a jacketedreaction vessel. To this, the required volume of 0.30 M Acetic acid andwater for injection (WFI) were added to maintain ˜0.1 M acetic acidconcentration. The pH of the solution was adjusted to 3.2±0.3 using 1 NNaOH or Glacial Acetic acid. The temperature of the reaction mixture wasincreased to 70±5° C. The reaction mixture was stirred at 70±5° C. for90-120 minutes. The reaction mixture was cooled down to 23±2° C. andneutralized (pH 7.0) by adding 1 M NaOH solution. The hydrolyzedpolysaccharide was purified by ultrafiltration/diafiltration against WFIusing 30K MWCO membranes. The solution was filtered through a 0.22 μmfilter and stored at 2 to 8° C. until oxidation. The molecular weight ofthe hydrolyzed polysaccharide was analyzed by SEC-MALLS to ensure thatthe molecular weight met the target range of 100 kDa to 350 kDa.

Partial Oxidation

In one experiment, the serotype 12F polysaccharide was mechanicallysized using pressure homogenization using a microfluidiser to reduce themolecular weight to approximately 100 kDa to 500 kDa. The sizedpolysaccharide was added to a reaction vessel at a concentration of 4.0mg/mL and mixed with bicarbonate/carbonate buffer (0.5 M NaHCO₃/0.05 MNa₂CO₃ buffer, pH 8.6) at a ratio of 1:1 v/v. To the stirred mixture wasadded ≤0.1 mol equivalent of TEMPO. The reaction was started by theaddition of 0.6 to 1.0 mol equivalent of NCS. The reaction mixture wasstirred at room temperature for 2 hours, after which the activatedpolysaccharide was purified by diafiltration, with WFI using a 30Kultrafiltration membrane. The purified polysaccharide was collected andthe degree of oxidation (DO) was determined by quantitative measurementsof aldehyde (using a 3-methyl-2-benothiazolinone hydrazone (MBTH) assay)and polysaccharide (using an anthrone assay).

In another experiment, the serotype 12F polysaccharide was hydrolyzed toreduce the molecular weight to a molecular weight of approximately 100kDa to 500 kDa. The serotype 12F polysaccharide was added to a reactionvessel and mixed with 0.5 M NaHCO₃/0.05 M Na₂CO₃ buffer (pH 8.6) at aratio of 1:1 v/v. To the stirred mixture was added 0.6 to 1.0 molarequivalents of NCS dissolved in WFI. The activation was initiated by theaddition of approximately 0.1 molar equivalents of TEMPO dissolved inWFI. The reaction mixture was stirred at room temperature for 2 hours,after which the activated polysaccharide was purified by diafiltrationwith WFI using a 30K ultrafiltration membrane. The purified activatedpolysaccharide was filtered through a 0.2 μm filter and stored at 4° C.before use.

The TEMPO/NCS mediated oxidations were also performed successfully insodium phosphate buffers of pH 6.5, 7.0, 7.5 and 8.0. In some activationexperiments a primary alcohol such as n-propanol was used to quench thereagents in order to avoid saccharide overoxidation. In another set ofexperiments the chemically hydrolysed polysaccharide was subjected tooxidation directly, without the ultrafiltration/diafiltrationpurification step.

Conjugation of Serotype 12F Oxidized Polysaccharide

In one experiment, the purified oxidized Serotype 12F polysaccharide wasadded to a reaction vessel followed by the addition of 0.5 M Sodiumphosphate buffer (pH 6.5) to a final buffer concentration of 0.1 M. Tothis solution, previously lyophilized CRM₁₉₇ was added and mixedthoroughly in order to obtain a homogenous solution. The pH was adjustedto 6.8 using diluted HCl or 1 N NaOH solution. This was followed by theaddition of 1.5 molar equivalents of NaCNBH₃. The reaction mixture wasstirred for 24 hours at room temperature (23° C.) and for 2.5 days at37° C. The reaction mixture was then diluted with 1×0.9% saline and theunreacted aldehyde groups were “capped” with 2 molar equivalents ofsodium borohydride. The capping reaction time was 3 hours.

In another experiment, the purified activated serotype 12F was added toa reaction vessel followed by the addition of 0.5 M sodium phosphatebuffer (pH 6.5) to a final buffer concentration of 0.1 M. To thissolution, previously lyophilized CRM₁₉₇ was added and mixed thoroughlyto obtain a homogenous solution. The pH was adjusted to 6.8 usingdiluted HCl or 1 N NaOH solution. This was followed by the addition of 3molar equivalents of NaCNBH₃. The reaction mixture was stirred for 24hours at 23° C. and for 48 hrs at 37° C. The reaction mixture was thendiluted with 1×0.9% saline and with stirring, the unreacted aldehydegroups were “capped” with 1 molar equivalent sodium borohydride NaBH₄.The capping reaction time was 3 hours.

In another experiment, the purified activated serotype 12F was added toa reaction vessel and mixed with CRM₁₉₇ solution. The mixture waslyophilized and the powder was dissolved in 0.1 M sodium phosphatebuffer (pH 6.8) to a final saccharide concentration of 5 mg/mL. Ifneeded the pH was adjusted to 6.8 using diluted HCl or 1N NaOH solution.This was followed by the addition of 3 molar equivalents NaCNBH₃.

The reaction mixture was stirred for 24 hours at 23° C. and for 48 hrsat 37° C. The reaction mixture was then diluted with 1×0.9% saline, theunreacted aldehyde groups were “capped” with 1 molar equivalent sodiumborohydride NaBH₄. The capping reaction time was 3 hours.

Conjugate Purification

The capped reaction mixture was filtered using a 5 μm filter and thenpurified using 100K MWCO ultra filtration membranes. The conjugate wasfirst diafiltered using 10 mM succinate/0.9% saline, pH 6.0 buffer. Thepurified conjugate was then filtered through 0.45/0.22 μm filters toobtain the bulk conjugate.

Degree of Oxidation

Successful oxidation of primary alcohols in the serotype 12Fpolysaccharide was achieved using the TEMPO/NCS system. The hydrolyzedSerotype 12F polysaccharides were oxidized to varying degrees ofoxidation (DO) levels by adjusting the amount of NCS cooxidant. Theeffect on DO by varying amounts of NCS using different polysaccharidebatches and molecular weights is shown in FIG. 9. Typically theoxidation reaction is complete in 2 hours as no significant change in DOwas observed after 2 hours.

Several serotytpe 12F conjugates were generated and characterized usingthe TEMPO/NCS oxidized polysaccharide. The results are summarized inTable 12.

TABLE 12 Pneumococcal Serotype 12F-CRM₁₉₇ conjugates Conjugate Batch12F-84A 12F-97B 12F-147C 12F-171D 12F-177-6E 12F-181F Oxidation Time 2 24 2 2 2 (hr) Degree of 12.0 6.0 9.6 12.0 11.5 11.5 Oxidation (DO) %Activated 80 71 70 89 86 86 Saccharide Yield Activated 137 155 170 190240 240 Saccharide MW by MALLS (kDa) Conjugation Lyo- Lyo- Lyo-CRMLyo-CRM Lyo-CRM Co-Lyo process CRM CRM Conjugate Results Saccharideyield 51.6 76.8 53.6 76.3 65.8 40.7 (%) Saccharide/Protein 1.2 0.9 1.01.1 1.4 0.9 Ratio % Free 24 10 17 20 23 14 Saccharide MW by SEC- 20503000 3600 1500 2400 2100 MALLS (kDa)

Example 8 Immunocienicity of Pn-Serotype 12F-CRM₁₉₇ Conjugates Using theTEMPO/NCS Oxidation Method

The opsonophagocytic activity (OPA) titers for serotype 12F-CRM₁₉₇conjugates in mice were determined in mice under standard conditions.OPA titers (geometric mean titer (GMT) with 95% confidence interval(CI)) at four and seven weeks are shown in Table 13, demonstrating thatthe serotype 12F-CRM₁₉₇ conjugate (Batch 12F-97B; also see Table 12 forcharacterization data of this conjugate) elicited OPA titers in a murineimmunogenicity model. The conjugate generated by the TEMPO-NCS was moreimmunogenic than the control conjugate (171B) generated from theperiodate oxidation.

TABLE 13 Immunogenicity of Serotype 12F-CRM₁₉₇ Conjugates Dose ConjugateSample 0.001 μg 0.01 μg 0.1 μg Periodate Oxidation (171B) Control 4 16172 TEMPO/NCS Oxidation (12F-97B) 40 417 880

Example 9 Evaluation of Pn-12F Glycoconjugates Stability

Comparison of the stability (at 25° C.) of the conjugates generated byperiodate oxidation vs. TEMPO/NCS oxidation (see FIG. 10) demonstratedthat the conjugate generated by the oxidation of the Pn-12Fpolysaccharides were relatively more stable. As shown in FIG. 10, anincrease in the free saccharide over time was observed for theglycoconjugate generated by the periodate oxidation of the Pn-12Fpolysaccharide at 25° C. In contrast, the glycoconjugate prepared usingthe TEMPO/NCS oxidation of the Pn-12F polysaccharide showed nosignificant trends for the free saccharide under similar conditions.

Example 10 Preparation of Serotype 15B Polysaccharide—CRM₁₉₇ Conjugate

Preparation of isolated Streptococcus pneumoniae serotype 15Bpolysaccharide Serotype 15B capsular polysaccharides can be obtaineddirectly from bacteria using isolation procedures known to one ofordinary skill in the art. The S. pneumoniae serotype 15B were grown ina seed bottle and then transferred to a seed fermentor. Once thetargeted optical density was reached, the cells were transferred to aproduction fermentor. The fermentation was broth was inactivated by theaddition of N-lauroyl sarcosine and purified by ultrafiltration anddiafiltration.

The purified S. pneumoniae serotype 15B polysaccharide was then sized byhigh pressure homogenization using a PANDA 2K® homogenizer (GEA NiroSoavi, Parma, Italy) to produce the isolated S. pneumoniae serotype 15Bpolysaccharide.

Preferably, the isolated S. pneumoniae serotype 15B capsularpolysaccharide obtained by the above process comprises at least 0.6 mMacetate per mM of serotype 15B capsular polysaccharide and has amolecular weight between 50 kDa and 500 kDa, preferably 150 kDa to 350kDa.

Oxidation of Isolated Streptococcus pneumoniae Serotype 15B CapsularPolysaccharide

Polysaccharide oxidation was carried out in 100 mM potassium phosphatebuffer (pH 6.0) by sequential addition of calculated amount of 500 mMpotassium phosphate buffer (pH 6.0) and WFI to give final polysaccharideconcentration of 2.0 g/L. If required, the reaction pH was adjusted topH 6.0, approximately. After pH adjustment, the reaction temperature wasadjusted to 23° C. Oxidation was initiated by the addition ofapproximately 0.25 molar equivalents of sodium periodate. The oxidationreaction was performed at 23° C. during 16 hrs, approximately.

Concentration and diafiltration of the activated polysaccharide wascarried out using 10K MWCO ultrafiltration cassettes. Diafiltration wasperformed against 20-fold diavolumes of WFI. The purified activatedpolysaccharide was then stored at 5° C. The purified activatedsaccharide was characterized inter alia by (i) saccharide concentrationby colorimetric assay; (ii) aldehyde concentration by colorimetricassay; (iii) Degree of Oxidation (iv) Molecular Weight by SEC-MALLS and(v) presence of O-acetyl and glycerol.

SEC-MALLS is used for the determination of the molecular weight ofpolysaccharides and polysaccharide-protein conjugates. SEC is used toseparate the polysaccharides by hydrodynamic volume. Refractive index(RI) and multi-angle laser light scattering (MALLS) detectors are usedfor the determination of the molecular weight. When light interacts withmatter, it scatters and the amount of scattered light is related to theconcentration, the square of the do/dc (the specific refractive indexincrements), and the molar mass of the matter. The molecular weightmeasurement is calculated based on the readings from the scattered lightsignal from the MALLS detector and the concentration signal from the RIdetector.

The degree of oxidation (DO=moles of sugar repeat unit/moles ofaldehyde) of the activated polysaccharide was determined as follows:

The moles of sugar repeat unit is determined by various colorimetricmethods, example by using Anthrone method. The polysaccharide is firstbroken down to monosaccharides by the action of sulfuric acid and heat.The Anthrone reagent reacts with the hexoses to form a yellow greencolored complex whose absorbance is read spectrophotometrically at 625nm. Within the range of the assay, the absorbance is directlyproportional to the amount of hexose present.

The moles of aldehyde also are determined simultaneously, using MBTHcolorimetric method. The MBTH assay involves the formation of an azinecompound by reacting aldehyde groups (from a given sample) with a3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). The excess3-methyl-2-benzothiazolone hydrazone oxidizes to form a reactive cation.The reactive cation and the azine react to form a blue chromophore. Theformed chromophore is then read spectroscopically at 650 nm.

Preferably, the activated S. pneumoniae serotype 15B capsularpolysaccharide obtained by the above process comprises at least 0.6 mMacetate per mM of serotype 15B capsular polysaccharide and has amolecular weight between 50 kDa and 500 kDa, preferably 150 kDa to 350kDa.

Conjugation of Activated S. pneumoniae Serotype 15B CapsularPolysaccharide with CRM₁₉₇

The conjugation process consisted in the following steps:

a) Compounding with sucrose excipient and lyophilization;

b) Reconstitution of the lyophilized activated polysaccharide andCRM₁₉₇;

c) Conjugation of activated polysaccharide to CRM₁₉₇ and capping; and

d) Purification of the conjugate

a) Compounding with Sucrose Excipient, and Lyophilization

The activated polysaccharide was compounded with sucrose to a ratio of25 grams of sucrose per gram of activated polysaccharide. The bottle ofcompounded mixture was then lyophilized. Following lyophilization,bottles containing lyophilized activated polysaccharide were stored at−20° C. Calculated amount of CRM₁₉₇ protein was shell-frozen andlyophilized separately. Lyophilized CRM₁₉₇ was stored at −20° C.

b) Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in anhydrousdimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide,an equal amount of anhydrous DMSO was added to lyophilized CRM₁₉₇ forreconstitution.

c) Conjugation and Capping

Reconstituted activated polysaccharide was combined with reconstitutedCRM₁₉₇ in the reaction vessel (input ratio: 0.8:1), followed by mixingthoroughly to obtain a clear solution before initiating the conjugationwith sodium cyanoborohydride. The final polysaccharide concentration inreaction solution is approximately 1 g/L. Conjugation was initiated byadding 1.0-1.5 MEq of sodium cyanoborohydride to the reaction mixtureand was incubated at 23° C. for 40-48 hrs. Conjugation reaction wasterminated by adding 2 MEq of sodium borohydride (NaBH₄) to capunreacted aldehydes. This capping reaction continued at 23° C. for 3 hrs

d) Purification of the Conjugate

The conjugate solution was diluted 1:10 with chilled 5 mM succinate-0.9%saline (pH 6.0) in preparation for purification by tangential flowfiltration using 100-300K MWCO membranes. The diluted conjugate solutionwas passed through a 5 μm filter and diafiltration was performed using 5mM succinate-0.9% saline (pH 6.0) as the medium. After the diafiltrationwas completed, the conjugate retentate was transferred through a 0.22 μmfilter.

The conjugate was diluted further with 5 mM succinate/0.9% saline (pH6), to a target saccharide concentration of approximately 0.5 mg/mL.Final 0.22 μm filtration step was completed to obtain theglycoconjugate.

Preferably, the conjugate obtained by the above process comprises atleast 0.6 mM acetate per mM of serotype 15B capsular polysaccharide, hasa molecular weight between 3,000 kDa and 20,000 kDa and has a degree ofconjugation between 2 and 6.

Example 11 Characterization of Glycoconjugate Comprising S. pneumoniaeSerotype 15B Capsular Polysaccharide Covlently Linked to a CRM₁₉₇

Conjugate 1 was prepared by the process of Example 10. Conjugates 2 and3 were prepared by a similar process using different amount of oxidizingagent. Conjugate 4 was prepared by a similar process except that thepurified serotype 15B capsular polysaccharide was not sized and wasactivated to a lower DO (higher oxidation level) and the conjugation wasperformed in aqueous medium. Conjugate 5 was prepared by a similarprocess except that the purified serotype 15B capsular polysaccharidewas sized by chemical hydrolysis and the conjugation was performed inaqueous medium. Conjugates 6 and 7 were prepared by a similar processexcept that the purified serotype 15B capsular polysaccharide was notsized.

The obtained conjugates were characterized and the results aresummarized in Table 14.

TABLE 14 Streptococcus pneumoniae serotype 15B capsular polysaccharide-CRM₁₉₇ conjugates Conjugate 1 2 3 4 5 6 7 Polysaccharide Sized SizedSized Native Hydrolyzed Native Native O-Acetyl; activated 0.69 0.69 0.691.01 0.66 0.76 N/A Polysaccharide (μmol acetate/μmol poly) Solventmedium DMSO DMSO DMSO Aqueous Aqueous DMSO DMSO Activated 11.4 5.8 9.74.8 8.8 5 12 Polysaccharide DO Activated 196 kDa 218 kDa 235 kDa 435 kDa270 kDa 431 kDa 460 kDa Polysaccharide MW Yield (%) 87.2 64 63.7 96.278.8 24.2 26.2 Saccharide Protein 0.68 0.65 0.71 1.22 1.29 0.9 1.5 RatioFree Saccharide (%) <5 <5 6.1 18.1 14.2 8.8 18 Conjugate MW, SEC- 61907090 7937 1766 1029 6293 4466 MALLS (kDa) O-Acetylation, 0.68 0.7 0.680.61 0.44 0.85 N/A Conjugate (μmol acetate/μmol poly) <0.3 K_(d) (%),SEC N/A 73 N/A N/A 62 N/A N/A Degree of Conj 3.7 3.9 4.1 N/A 3.4 N/A N/A(AAA); Modified Lys % O-Acetyl Retained 99% 100% 99.5% 60% 67% 100% N/Ain Conjugate N/A = not available

The percentage of free polysaccharide is measured by a procedureutilizing aluminum hydroxide gel to bind protein and covalently boundsaccharide for removal by centrifugation. Samples are mixed withphosphate buffered aluminum hydroxide gel and centrifuged. Boundsaccharide is pelleted with the gel and free saccharide remains in thesupernatant. The resulting supernatant and controls samples arequantitated by appropriate colorimetric assays to determine thepercentage of free saccharide and to confirm sufficient removal ofprotein and recovery of saccharide.

For the amino acid analysis the polysaccharide-protein sample is firsthydrolyzed into its individual components as free amino acids, using 6 Nhydrochloric acid (HCl) hydrolysis under vacuum and heat (160° C. for 15minutes). After hydrolysis, the samples are analyzed using Amino AcidAnalyzer. The individual amino acids are separated through ion exchangechromatography using a step gradient of sodium citrate buffer withtemperature and flow rate changes. After separation, the amount of eachamino acid residual is quantitatively determined using a postcolumnninhydrin coupling detection system. In this system, the ninhydrin ismixed with the column eluate in the postcolumn reactor system and themixture passed into the photometer. The reaction of ninhydrin witheluated amino acids yields a purple compound that absorbs maximally at570 nm. This absorbance is a linear response (function) of the amount ofα-amino groups present and this reaction provides quantitativecolorimetric assay for all organic compounds with α-amino groups. In thereaction with imino acids such as proline and hydroxylproline, which donot have free amino group, a bright yellow compound is generated andmonitored at 440 nm. The peak areas for each amino acid are calculatedusing both 570 nm and 440 nm wavelength outputs.

The yield is calculated as follows: (amount of polysaccharide in theconjugate×100)/amount of activated polysaccharide.

Conjugates (4 and 5) generated using an aqueous medium demonstratedsignificant loss in O-acetyl levels. Conjugates generated in DMSOsolvent, using native polysaccharide without MW sizing (6 and 7) did notdemonstrate loss in O-acetyl levels. However, the conjugate yields werevery poor in addition to poor filterability characteristics. Conjugatesgenerated in DMSO using polysaccharides that were sized by high pressurehomogenization (1, 2 and 3) had high yield and better filterabilitycharacteristics with significant preservation of O-acetyl levels. Theseconjugates also had very low levels of free polysaccharides.

Example 12 Opsonophagocytic Activity (OPA) Assay Using Pn-Serotype15B-CRM₁₉₇ Conjugates

The immunogenicity of the S. pneumoniae serotype 15B conjugates of theinvention can be assessed using the OPA assay described below.

Groups of 30 6-7 week old female Swiss Webster mice were immunized with0.001 μg, 0.01 μg, or 0.1 μg of test conjugates via the subcutaneousroute on week 0. The mice were boosted with the same dose of conjugateon week 3 and then bled at week 4.

Serotype-specific OPAs were performed on week 4 sera samples.

OPAs are used to measure functional antibodies in murine sera specificfor S. pneumoniae serotype 15B. Test serum is set up in assay reactionsthat measure the ability of capsular polysaccharide specificimmunoglobulin to opsonize bacteria, trigger complement deposition,thereby facilitating phagocytosis and killing of bacteria by phagocytes.The OPA titer is defined as the reciprocal dilution that results in a50% reduction in bacterial count over control wells without test serum.The OPA titer is interpolated from the two dilutions that encompass this50% killing cut-off.

OPA procedures were based on methods described in Hu et al. (2005) ClinDiagn Lab Immunol12 (2):287-295 with the following modifications. Testserum was serially diluted 2.5-fold and added to microtiter assayplates. Live serotype 15B target bacteria were added to the wells andthe plates were shaken at 37° C. for 30 minutes. Differentiated HL-60cells (phagocytes) and baby rabbit serum (3- to 4-week old, PEL-FREEZ®,6.25% final concentration) were added to the wells, and the plates wereshaken at 37° C. for 45 minutes. To terminate the reaction, 80 μL of0.9% NaCl was added to all wells, mixed, and a 10 μL aliquot weretransferred to the wells of MULTISCREEN® HTS HV filter plates(MILLIPORE®) containing 200 μL of water. Liquid was filtered through theplates under vacuum, and 150 μL of HYSOY® medium was added to each welland filtered through. The filter plates were then incubated at 37° C.,5% CO₂ overnight and were then fixed with Destain Solution (Bio-RadLaboratories, Inc., Hercules, Calif.). The plates were then stained withCoomassie Blue and destained once. Colonies were imaged and enumeratedon a Cellular Technology Limited (CTL) (Shaker Heights, Ohio)IMMUNOSPOT® Analyzer. Raw colony counts were used to plot kill curvesand calculate OPA titers.

The immunogenicity of conjugates 1 and 2 has been tested according tothe above mentioned assay. One additional conjugate and an unconjugatednative S. pneumoniae serotype 15B capsular polysaccharide (unconjugatedPS) were also tested in the same assay:

Conjugate 9 was prepared by conjugation of native (i.e., not sized)serotype 15B capsular polysaccharide to CRM₁₉₇ by reductive amination inaqueous solution. The results are shown at Table 15.

TABLE 15 OPA Titers of Animal Testing using Serotype 15B-CRM₁₉₇Conjugates OPA GMT (95% CI) 0.001 μg 0.01 μg 0.1 μg Conjugate 1 485(413, 569) 804 (565, 1145) 1563 (1048, 2330) Conjugate 2 556 (438, 707)871 (609, 1247) 1672 (1054, 2651) Conjugate 9 395 (329, 475) 856 (627,1168) 1802 (1108, 2930) Unconjugated — —  698 (466, 1045) PS

As shown in the Table 15 above, conjugates 1 and 2, when administered tomice, generated antibodies capable of opsonizing S. pneumoniae serotype15B, triggering complement deposition, thereby facilitating phagocytosisand killing of bacteria by phagocytes. In addition, despite their lowermolecular weight, they also exhibited similar level of immunogenicity ascompared to conjugate 9 which has not been sized.

Example 13 Preparation of Serotype 22F Polysaccharide—CRM₁₉₇ Conjugate

Preparation of Isolated S. pneumoniae Serotype 22F Polysaccharide

The S. pneumoniae serotype 22F were grown in a seed bottle and thentransferred to a seed fermentor. Once the targeted optical density wasreached, the cells were transferred to a production fermentor. Thefermentation was broth was inactivated by the addition of N-lauroylsarcosine and purified by ultrafiltration and diafiltration.

The purified S. pneumoniae serotype 22F polysaccharide was sized by highpressure homogenization using a PANDA 2K® homogenizer (GEA Niro Soavi,Parma, Italy) to produce the isolated S. pneumoniae serotype 22Fpolysaccharide

Oxidation of Isolated S. pneumoniae Serotype 22F Capsular Polysaccharide

Oxidation of polysaccharide was carried out in 100 mM potassiumphosphate buffer (pH 5.8) obtained by sequential addition of calculatedamount of 500 mM potassium phosphate buffer (pH 5.8) and WFI to givefinal polysaccharide concentration of 2.0 g/L. If required, the reactionpH was adjusted to 5.8, approximately. After pH adjustment, the reactiontemperature was lowered to 5° C. Oxidation was initiated by the additionof 0.10 molar equivalents (MEq) of sodium periodate. The targetoxidation reaction time is 16 hrs at 5° C.

The oxidation reaction was quenched with 2 MEq of 2,3-butanediol undercontinuous stirring at 5° C. for 1-2 hrs.

Concentration and diafiltration of the activated polysaccharide wascarried out using 100K MWCO ultrafiltration cassettes. Diafiltration wasperformed against 35-fold diavolume of WFI. The purified activatedpolysaccharide was stored at 5° C. The purified activated saccharide ischaracterized inter alia by (i) Molecular Weight by SEC-MALLS (ii)presence of O-acetyl and (iii) Degree of Oxidation.

SEC-MALLS is used for the determination of the molecular weight ofpolysaccharides and polysaccharide-protein conjugates. SEC is used toseparate the polysaccharides by hydrodynamic volume. Refractive index(RI) and multi-angle laser light scattering (MALLS) detectors are usedfor the determination of the molecular weight. When light interacts withmatter, it scatters and the amount of scattered light is related to theconcentration, the square of the do/dc (the specific refractive indexincrements), and the molar mass of the matter. The molecular weightmeasurement is calculated based on the readings from the scattered lightsignal from the MALLS detector and the concentration signal from the RIdetector.

The degree of oxidation (DO=moles of sugar repeat unit/moles ofaldehyde) of the activated polysaccharide was determined as follows:

The moles of sugar repeat unit is determined by various colorimetricmethods, for example by using Anthrone method. The polysaccharide isfirst broken down to monosaccharides by the action of sulfuric acid andheat. The Anthrone reagent reacts with the hexoses to form a yellowgreen colored complex whose absorbance is read spectrophotometrically at625 nm. Within the range of the assay, the absorbance is directlyproportional to the amount of hexose present.

The moles of aldehyde also are determined simultaneously, using MBTHcolorimetric method. The MBTH assay involves the formation of an azinecompound by reacting aldehyde groups (from a given sample) with a3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). The excess3-methyl-2-benzothiazolone hydrazone oxidizes to form a reactive cation.The reactive cation and the azine react to form a blue chromophore. Theformed chromophore is then read spectroscopically at 650 nm.

Conjugation of Activated S. pneumoniae Serotype 22F Polysaccharide withCRM₁₉₇

The conjugation process consisted in the following steps:

a. Compounding with sucrose excipient, and lyophilization;

b. Reconstitution of the lyophilized polysaccharide and CRM₁₉₇;

c. Conjugation of activated polysaccharide to CRM₁₉₇ and capping; and

d. Purification of the conjugate

a. Compounding with Sucrose and Lyophilization

The activated polysaccharide was compounded with sucrose (50% w/v inWFI) to a ratio of 25 grams of sucrose per gram of activatedpolysaccharide. The bottle of compounded mixture was then lyophilized.Following lyophilization, bottles containing lyophilized activatedpolysaccharide were stored at −20° C. Calculated amount of CRM₁₉₇protein (target S/P input ratio=1) was shellfrozen and lyophilizedseparately. Lyophilized CRM₁₉₇ was stored at −20° C.

b. Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in anhydrousdimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide,an equal amount of anhydrous DMSO was added to lyophilized CRM₁₉₇ forreconstitution.

c. Conjugation of Activated Polysaccharide to CRM₁₉₇ and Capping

Reconstituted CRM₁₉₇ (in DMSO) was combined in the conjugation reactionvessel with the reconstituted activated polysaccharide. The finalpolysaccharide concentration in reaction solution is 1 g/L. Conjugationwas initiated by adding 1.5 MEq of sodium cyanoborohydride to thereaction mixture and the reaction was incubated at 23° C. for 20 hrs.Termination of conjugation reaction is done by adding 2 MEq of sodiumborohydride. The capping reaction was incubated at 23° C. for 3 hrs.

d. Purification of Conjugate

The conjugate solution was diluted 1:5 with chilled 5 mM succinate-0.9%saline (pH 6.0) in preparation for purification by tangential flowfiltration using 100K MWCO membranes and a 20× diafiltration wasperformed using 5 mM succinate-0.9% saline (pH6.0) as the medium. Afterthe diafiltration was completed, the conjugate retentate was furtherdiluted, filtered through a 0.22 μm filter and stored at 2-8° C.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and Meq of sodium cyanoborohydride).Characterization for representative Pn-22F glycoconjugates to CRM₁₉₇ isprovided in Table 16

TABLE 16 Pneumococcal Serotype 22F-CRM₁₉₇ conjugates Batch 1 2 3 4 5 6 78 9 10 Degree of 12.6 19.5 17.2 14.0 12.4 14.9 11.1 14.6 14.4 13.7Oxidation (D.O) Activated 540 697 864 92 866 631 614 639 709 416Saccharide MW by MALLS (kDa) Conjugate Results Saccharide/Protein 0.750.87 2 0.8 0.8 0.4 1.9 0.8 0.65 1.0 Ratio O—Ac (%) 105 100 N/A N/A N/AN/A N/A N/A N/A N/A % Free Saccharide <5 2 15.5 35 <5 <5 33 <5 <5 8 MWby SEC- 2787 1668 2194 1419 5039 10450 1577 3911 3734 4453 MALLS (kDa)N/A = not available

The % O-Acetyl (preserved) level in the final conjugate was calculatedfrom the ratio of the O-Acetyl content of the conjugate (μmol O-Acetylper μmol of the serotype 22F saccharide repeat unit) relative to theO-Acetyl content of the polysaccharide (μmol O-Acetyl per μmol of theserotype 22F saccharide repeat unit).

The immunogenicity of the conjugates obtained above have been assessedusing the opsonophagocytic assay (OPA) described below.

Groups of thirty 6-7 week old female Swiss Webster mice were immunizedwith 0.001 μg, 0.005 μg or 0.01 μg of test conjugates via thesubcutaneous route on week 0. The mice were boosted with the same doseof conjugate on week 3 and then bled at week 4. Serotype-specific OPAswere performed on week 4 sera samples.

Opsonophagocytic activity (OPA) assays are used to measure functionalantibodies in murine sera specific for S. pneumonia serotype 22F. Testserum is set up in assay reactions that measure the ability of capsularpolysaccharide specific immunoglobulin to opsonize bacteria, triggercomplement deposition, thereby facilitating phagocytosis and killing ofbacteria by phagocytes. The OPA titer is defined as the reciprocaldilution that results in a 50% reduction in bacterial count over controlwells without test serum. The OPA titer is interpolated from the twodilutions that encompass this 50% killing cut-off.

OPA procedures were based on methods described in Hu et al. (2005) ClinDiagn Lab Immunol 12(2):287-295 with the following modifications. Testserum was serially diluted 2.5-fold and added to microtiter assayplates. Live serotype 22F target bacterial strains were added to thewells and the plates were shaken at 25° C. for 30 minutes.Differentiated HL-60 cells (phagocytes) and baby rabbit serum (3- to4-week old, PEL-FREEZ®, 12.5% final concentration) were added to thewells, and the plates were shaken at 37° C. for 45 minutes. To terminatethe reaction, 80 μL of 0.9% NaCl was added to all wells, mixed, and a 10μL aliquot were transferred to the wells of MULTISCREEN® HTS HV filterplates (MILLIPORE®) containing 200 μL of water. Liquid was filteredthrough the plates under vacuum, and 150 μL of HYSOY® medium was addedto each well and filtered through. The filter plates were then incubatedat 37° C., 5% CO₂ overnight and were then fixed with Destain Solution(Bio-Rad Laboratories, Inc., Hercules, Calif.). The plates were thenstained with Coomassie Blue and destained once. Colonies were imaged andenumerated on a Cellular Technology Limited (CTL) (Shaker Heights, Ohio)IMMUNOSPOT® Analyzer. Raw colony counts were used to plot kill curvesand calculate OPA titers.

The Opsonophagocytic activity (OPA) titers for Serotype 22F-CRM₁₉₇conjugates were determined as mentioned above. OPA titers (geometricmean titer (GMT) with 95% confidence interval (CI)) at four weeks atdifferent doses are shown in Tables 17 and 18, (two separateexperiments) demonstrating that the serotype 22F conjugate (Batches 1-7;also see Table 16 for characterization data of these conjugates)elicited OPA titers in a murine immunogenicity model.

TABLE 17 Immunogenicity of Serotype 22F-CRM₁₉₇ Conjugates OPA GMT (95%CI) Sample No. 0.001 μg 0.005 μg 0.01 μg 1 86 (45, 165) 597 (285, 1252)2519 (1409, 4504) 2 98 (51, 191) 782 (410, 1492) 2236 (1319, 3790) 3 35(18, 69) 250 (122, 512)  509 (273, 950)

TABLE 18 Immunogenicity of Serotype 22F-CRM₁₉₇ Conjugates OPA GMT (95%CI) Sample No. 0.001 μg 0.01 μg 4  37 (18, 76) 3383 (1911, 5987) 5  45(20, 103) 1773 (1072, 2931) 6 235 (108, 513) 4335 (3018, 6226) 7  10 (7,13)  252 (138, 457)

Example 14 Preparation of Pn-11A Conjugates to CRM₁₉₇

Preparation of Pn-11A RAC Glycoconjucates

The frozen sized polysaccharide stored in de-ionized water or 25 mMpotassium phosphate buffer (pH 6.0) was thawed at 5° C.

Oxidation of Polysaccharide

Polysaccharide oxidation was carried out in 100 mM potassium phosphatebuffer (pH 6.0) by addition of 500 mM potassium phosphate buffer (pH6.0) and WFI to give final polysaccharide concentration of 2.0 g/L.Oxidation reaction was carried out at 23° C. Oxidation was initiated bythe addition of sodium periodate. The agitation rate ranges from 100-140rpm.

Purification of Activated 11A Polysaccharide

Concentration and diafiltration of the activated polysaccharide wascarried out using ultrafiltration cassettes. Diafiltration was performedagainst 20-fold diavolume of WFI. After 0.22 μm filtration, the purifiedactivated polysaccharide was stored at 5° C.

Conjugation Process Description

The conjugation process consisted in the following steps:

-   -   a. Shell freezing and lyophilization of CRM₁₉₇ protein;    -   b. Reconstitution of the activated polysaccharide and CRM₁₉₇;    -   c. Conjugation of activated polysaccharide to CRM₁₉₇; and    -   d. Purification and dilution of the conjugate

a. Shell Freezing and Lyophilization of CRM₁₉₇ Protein

CRM₁₉₇ protein was shell-frozen and lyophilized.

b. Reconstitution of Activated Polysaccharide and CRM₁₉₇ Protein

Activated polysaccharide solution (˜10 g/L) was charged into reactorfollowed by addition of calculated amount 0.5 N sodium phosphate buffer(pH 7.2). Under stirring, lyophilized CRM₁₉₇ was added and the reactionmixture was stirred for 2-4 hours in order to reach complete dissolutionof CRM₁₉₇.

c. Conjugation and Capping

Conjugation was initiated by adding cyanoborohydride. The reactionmixture was incubated at 23° C. for 72-96 hrs. Termination ofconjugation reaction was done by adding 0.5×WFI followed by 2 MEq ofsodium borohydride. This capping reaction was kept at 23° C. for 3-4hrs.

d. Dilution and Initial Purification of Conjugate

The conjugate solution was diluted 1:5 (reaction volume) with 0.15 Nsodium phosphate buffer (pH 8.0) in preparation for purification bytangential flow filtration (TFF). Diluted conjugate was mixed in thedilution vessel and then passed through a 5 μm filter. The filteredconjugate solution was then concentrated down to 1-2 g/L. A two-stepsdiafiltration process was performed. In step one, TFF was carried outusing 30× (diafiltration volume) of 0.15 N sodium phosphate buffer (pH8.0) followed by 20× of 5 mM succinate-0.9% NaCl (pH6.0). After theinitial diafiltration was completed, the conjugate retentate wastransferred through a 0.45 μm filter into a collection tank.

Final Diafiltration of Conjugate

The final purification step was a 20× diafiltration with 5 mMsuccinate-0.9% NaCl, pH 6.0 medium using regenerated cellulosemembranes.

Dilution of the Monovalent Bulk Conjugate (MBC)

The conjugate was diluted further with 5 mM succinate/0.9% NaCl, pH 6,to a target saccharide concentration of 0.5 mg/mL. Final 0.22 μmfiltration step was completed to prepare the monovalent bulk conjugate(MBC) product for formulation.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and Meq of sodium cyanoborohydride).Characterization for representative Pn-11A glycoconjugates to CRM₁₉₇ isprovided in Table 19 (batches 1 to 5).

Preparation of Pn-11A Glycoconjugates using RAC/DMSO

Oxidized polysaccharide was prepared and purified as described above(see Preparation of Pn-11A RAC Glycoconjugates).

Conjugation via Reductive Amination in DMSO (RAC/DMSO)

Conjugation of 11A through RAC/DMSO consisted of the following steps:

-   -   a. Compounding with sucrose, shell freezing and lyophilization;    -   b. Reconstitution of the lyophilized polysaccharide and CRM₁₉₇;    -   c. Conjugation of activated polysaccharide to CRM₁₉₇; and    -   d. Purification and dilution of the conjugate.

a. Compounding with Sucrose, Shell Freezing and Lyophilization

The activated polysaccharide prepared from sized polysaccharide wascompounded with sucrose (50% w/v in WFI) to a ratio of 25 grams ofsucrose per gram of activated polysaccharide. The components were mixedthe shell-frozen bottle of compounded mixture was then lyophilized.CRM₁₉₇ protein was shell-frozen and lyophilized separately.

b. Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in DMSO at 2mg/mL concentration. Upon the complete dissolution of polysaccharide,DMSO was added to lyophilized CRM₁₉₇ for reconstitution

c. Conjugation and Capping

Reconstituted CRM₁₉₇ (in DMSO) was combined in the conjugation reactionvessel with the reconstituted activated polysaccharide. The finalpolysaccharide concentration in reaction solution is 1 g/L. Conjugationwas initiated by adding cyanoborohydride to the reaction mixture and wasincubated at 23° C. for 22 hours. Termination of conjugation reaction isdone by adding 2 MEq of sodium borohydride. This capping reaction waskept at 23° C. for 3-4 hrs.

d. Purification and Dilution of the Conjugate

The conjugate solution was purified and diluted using a similar processas described above.

Several conjugates were obtained using the above described process byvarying different parameters (e.g., saccharide-protein input ratio,reaction concentration and Meq of sodium cyanoborohydride).Characterization for representative Pn-11A glycoconjugates to CRM₁₉₇obtained by the above process is provided at Table 19 (batches 6 to 8).

TABLE 19 Pneumococcal Serotype 11A-CRM₁₉₇ conjugates Batch 1 2 3 4 5 6 78 Activated Saccharide 207 129 103 199 183 232 113 113 MW by MALLS (kDa)Conjugate Results Saccharide/Protein 1.24 1.09 1.32 1.47 1.31 1 0.780.68 Ratio Acetate (mol/mol PS) 2.72 2.89 2.72 3.2 3.13 N/A N/A N/AGlycerol (mol/mol PS)* 0.62 0.68 0.75 0.51 0.41 N/A N/A N/A MW bySEC-MALLS 3224 837 623 827 994 12200 6543 15730 (kDa) N/A = notavailable *Glycerol was quantitated by High Performance Anion ExchangeChromatography with Pulsed Amperometric Detection (HPAEC-PAD) after itsrelease from the polysaccharide by hydrofluoric acid (HF).

The overall data generated from conjugates prepared by the abovereductive amination processes demonstrated that it allowed preparingconjugates with good conjugation yield, low % free saccharide and withgood stability.

The immunogenicity of the conjugates obtained above have been assessedusing the opsonophagocytic assay (OPA) described below.

Groups of thirty 6-7 week old female Swiss Webster mice were immunizedwith 0.001 μg, 0.005 μg, 0.01 μg, or 0.1 μg of test conjugates via thesubcutaneous route on week 0. The mice were boosted with the same doseof conjugate on week 3 and then bled at week 4. Serotype-specific OPAswere performed on week 4 sera samples.

Opsonophagocytic activity (OPA) assays are used to measure functionalantibodies in murine sera specific for S. pneumonia serotype 11A. Testserum is set up in assay reactions that measure the ability of capsularpolysaccharide specific immunoglobulin to opsonize bacteria, triggercomplement deposition, thereby facilitating phagocytosis and killing ofbacteria by phagocytes. The OPA titer is defined as the reciprocaldilution that results in a 50% reduction in bacterial count over controlwells without test serum. The OPA titer is interpolated from the twodilutions that encompass this 50% killing cut-off.

OPA procedures were based on methods described in Hu et al. (2005) ClinDiagn Lab Immunol12 (2):287-295 with the following modifications. Testserum was serially diluted 2.5-fold and added to microtiter assayplates. Live serotype 22F target bacterial strains were added to thewells and the plates were shaken at 25° C. for 30 minutes.Differentiated HL-60 cells (phagocytes) and baby rabbit serum (3- to4-week old, PEL-FREEZ®, 12.5% final concentration) were added to thewells, and the plates were shaken at 37° C. for 60 minutes. To terminatethe reaction, 80 μL of 0.9% NaCl was added to all wells, mixed, and a 10μL aliquot were transferred to the wells of MULTISCREEN® HTS HV filterplates (MILLIPORE®) containing 200 μL of water. Liquid was filteredthrough the plates under vacuum, and 150 μL of HYSOY® medium was addedto each well and filtered through. The filter plates were then incubatedat 37° C., 5% CO₂ overnight and were then fixed with Destain Solution(Bio-Rad Laboratories, Inc., Hercules, Calif.). The plates were thenstained with Coomassie Blue and destained once. Colonies were imaged andenumerated on a Cellular Technology Limited (CTL) (Shaker Heights, Ohio)IMMUNOSPOT® Analyzer. Raw colony counts were used to plot kill curvesand calculate OPA titers.

The Opsonophagocytic activity (OPA) titers for serotype 11A-CRM₁₉₇conjugates in mice were determined as mentioned above. OPA titers(geometric mean titer (GMT) with 95% confidence interval (CI)) at fourweeks at different doses are shown in Table 20, demonstrating that theserotype 11A conjugate (Batches 2-4 and 8; also see Table 19 forcharacterization data of these conjugates) elicited OPA titers in amurine immunogenicity model.

TABLE 20 Immunogenicity of Serotype 11A-CRM₁₉₇ Conjugates OPA GMT (95%CI) Batch No. 0.001 μg 0.01 μg 0.1 μg 2 326 (260, 408) 1391 (794, 2437)4366 (3063, 6223) 3 389 (316, 478) 1113 (690, 1795) 5527 (3698, 8260) 4192 (149, 248)  926 (661, 1298) 2800 (1975, 3970) 8 303 (224, 411) 1099(624, 1935) 3861 (2629, 5669)

Example 15 Formulation of a 16-Valent Pneumococcal Conjugate Vaccine

A 16-valent conjugates composition comprising glycoconjugates from S.pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F,22F, 23F and 33F (16vPnC) all individually conjugated to CRM₁₉₇ wasformulated.

Glycoconjugates from S. pneumoniae from serotypes 15B, 22F and 33F wereproduced as disclosed above and S. pneumoniae glycoconjugates fromserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F wereproduced as disclosed in WO 2006/110381.

The required volumes of bulk concentrates were calculated based on thebatch volume and the bulk saccharide concentrations. The formulated bulkvaccine was prepared by adding the required volume of NaCl/succinatebuffer (pH 5.8) to obtain a final target buffer concentration ofsuccinate 5.0 mM and 150 mM NaCl. Polysorbate 80 to a finalconcentration of 0.02% and the 16 pneumococcal conjugates were added.The preparation was filtered through a 0.2 μm Millipore PES membrane,followed by the addition of AlPO4. The formulation was mixed to allowfor binding and to achieve homogeneity.

The formulation was then filled into glass syringes to deliver a dosevolume of 0.5 mL.

The final dosage form consisted in 2.2 μg of each of glycoconjugatesfrom S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 15B, 18C, 19A,19F, 22F, 23F and 33F individually conjugated to CRM₁₉₇, 4.4 μg ofglycoconjugate from S. pneumoniae serotype 6B, 5 mM succinate buffer pH5.8, 0.02 PS80, 150 mM NaCl and 0.25 mg/mL aluminum as AlPO₄ for a doseof 0.5 mL. CRM₁₉₇, content was about 38 μg for a dose of 0.5 mL.

Example 16 Formulation of a 20-Valent Pneumococcal Conjugate Vaccine

A 20 valent conjugates composition comprising glycoconjugates from S.pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14,15B, 18C, 19A, 19F, 22F, 23F and 33F (20vPnC) all individuallyconjugated to CRM₁₉₇ was formulated.

Glycoconjugates from S. pneumoniae from serotypes 8, 10A, 11A, 12F, 15B,22F and 33F were produced as disclosed above and S. pneumoniaeglycoconjugates from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A,19F and 23F were produced as disclosed in WO 2006/110381.

The required volumes of bulk concentrates were calculated based on thebatch volume and the bulk saccharide concentrations. The formulated bulkvaccine was prepared by adding the required volume of NaCl/succinatebuffer (pH 5.8) to obtain a final target buffer concentration ofsuccinate 5.0 mM and 150 mM NaCl. Polysorbate 80 to a finalconcentration of 0.02% and the 20 pneumococcal conjugates are added. Thepreparation was filtered through a 0.2 μm Millipore PES membrane,followed by the addition of AlPO₄. The formulation was mixed well toobtain maximum binding of the conjugates to the aluminum.

The formulation is then filled into glass syringes to deliver a dosevolume of 0.5 mL.

The final dosage form consisted in 2.2 μg of each of glycoconjugatesfrom S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F,14, 15B, 18C, 19A, 19F, 22F, 23F and 33F individually conjugated toCRM₁₉₇, 4.4 μg of glycoconjugate from S. pneumoniae serotype 6B, 5 mMsuccinate buffer pH 5.8, 0.02 PS80, 150 mM NaCl and 0.25 mg/mL aluminumas AlPO4 for a dose of 0.5 mL. CRM₁₉₇, content was about 46 μg for adose of 0.5 mL.

Example 17 Immunogenicity of a 16-Valent Immunogenic Composition

The immunogenicity of the 16-valent immunogenic composition (see Example15) was assessed in Rabbits using multiplexed direct Lumineximmunoassays (dLIAs) to measure serotype-specific IgG concentrations insera and serotype-specific OPAs.

Groups of ten 2.5 kg to 3.5 kg female New Zealand white rabbits wereimmunized with the proposed human clinical dose (2.2 μg of conjugateexcept serotype 6B which was at 4.4 μg; plus 0.1 mg aluminum as AlPO₄)via the intramuscular route on week 0. The rabbits were boosted with thesame dose of conjugate vaccine on week 2 and then bled at week 4.Serotype-specific dLIAs and OPAs were performed on week 0 and week 4sera samples.

To quantify the total polysaccharide binding antibody (IgG) specific toeach pneumococcal polysaccharide (PnPS), rabbit sera were evaluated intwo direct Luminex immunoassays (dLIAs; 13-plex dLIA, PREVNAR 13®serotypes and 7-plex dLIA, additional serotypes). The 13-plex assaymeasures anti-PnPS antibodies specific to the 13 serotypes included inthe 13-valent pneumococcal conjugate (PnC) vaccine (1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F, and 23F) and the 7-plex assay measuresanti-PnPS antibodies to the additional serotypes (15B, 22F, 33F). Eachassay contains a combination of 13 or 7 spectrally distinct magneticmicrospheres coupled to PnPS conjugates (PnPS-PLL conjugates: PnPSconjugated to poly-L-Lysine).

Briefly, reference standard, controls and test sera were firstpre-adsorbed with two Pn absorbents; CWPS1 (cell wall polysaccharidefrom PnA containing C-polysaccharide) and CWPS2 (CWP from acapsular S.pneumoniae serotype 2) to block non-specific antibodies from binding tothe PnPS coating antigen. Following preadsorption, the PnPS-coupledmicrospheres were incubated with appropriately diluted referencestandard serum, controls or rabbit test sera. After incubation, eachmixture was washed and an R-Phycoerythrin-conjugated goat anti-rabbitIgG secondary antibody was added. Fluorescent signals (expressed asmedian fluorescence intensities (MFIs)) were measured using a Bio-Plexreader and correlated to the amount of bound PnPS-specific IgG. Valuesfor test sera are reported as (Units/mL, U/mL).

Serotype-specific OPAs were performed as described above. The OPA titeris the reciprocal of the highest serum dilution resulting in 50%reduction in the number of bacterial colony forming units (CFUs) whencompared to the control without serum (defined as the background CFU).The titer is interpolated from the two dilutions that encompass this 50%killing cut-off.

TABLE 21 16vPnC Total IgG Concentrations and OPA Titers Total IgG (PndLIA) Opsonophagocytic Antibody (OPA) Wk 0 Wk 4 IgG GMC Wk Wk Wk 4 OPAGMT GMC GMC Wk 4 95% Ratio 0 4 95% CI Ratio Serotype (μg/ml) (μg/ml) CI(LCI-UCI) Wk4:Wk0 GMT GMT (LCI-UCI) Wk 4:Wk 0  1 0.08 28 17-44 369 4 87 55-139 22  3 0.08 88  60-128 1062 4 214 151-304 54  4 0.08 30 14-67 4024 934  551-1583 233  5 0.08 34 18-64 449 4 368 232-584 87  6A 0.03 46 15-142 1835 4 3026 1607-5696 756  6B 0.08 89  33-241 1182 4 6156 3043-12453 1539  7F 0.01 50 31-78 3969 6 2917 2013-4227 528  9V 0.03 2415-38 881 5 613 426-883 112 14 0.08 28 20-39 368 19 449 331-610 24 18C0.05 79  45-139 1587 4 1847 1003-3401 462 19A 0.08 120  71-205 1605 41410  851-2336 352 19F 0.08 156  96-255 2083 4 3207 1783-5771 802 23F0.05 33 13-84 668 4 997  487-2042 249 15B 0.05 54 40-71 1073 6 741 514-1069 116 22F 0.08 158  95-262 2103 5 1078  661-1756 211 33F 0.10 11 6-20 115 49 1337  829-2154 27 Abbreviations: GMC, geometric meanconcentration; CI, confidence interval; LCI, lower confidence interval;UCI, upper confidence interval.

Results showed a significant increase in serotype-specific IgG andfunctional OPA antibody responses following two immunizations with16vPnC (Table 21). Serum IgG levels increased more than 2-logs abovebaseline. Similarly, a robust functional OPA antibody response waselicited with a minimum of a 22-fold increase in OPA GMT above baseline.Pre-immune sera (Wk 0) showed undetectable levels of PnPS-specific IgGand functional OPA antibody for the majority of the 16 v Pn serotypeswith the exception of serotypes 14 and 33F. Low level OPA titers werepresent for these serotypes but these baseline responses did notadversely affect the antibody response following vaccination.

Example 18 Immunogenicity of a 20-Valent Immunogenic Composition

The immunogenicity of the 20-valent immunogenic composition (as preparedat example 16) was assessed in rabbits using multiplexed direct Lumineximmunoassays (dLIAs) to measure serotype-specific IgG concentrations insera and serotype-specific OPAs.

Groups of ten 2.5 kg to 3.5 kg female New Zealand white rabbits wereimmunized with the proposed human clinical dose (2.2 μg of conjugateexcept serotype 6B which was at 4.4 μg; plus 0.1 mg aluminum as AlPO₄)via the intramuscular route on week 0. The rabbits were boosted with thesame dose of conjugate vaccine on week 2 and then bled at week 4.Serotype-specific dLIAs and OPAs were performed on week 0 and week 4sera samples.

To quantify the total polysaccharide binding antibody (IgG) specific toeach pneumococcal polysaccharide (PnPS), rabbit sera were evaluated intwo direct Luminex immunoassays (dLIAs; 13-plex dLIA, PREVNAR 13®serotypes and 7-plex dLIA, additional serotypes). The 13-plex assaymeasures anti-PnPS antibodies specific to the 13 serotypes included inthe 13-valent pneumococcal conjugate (PnC) vaccine (1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F, and 23F) and the 7-plex assay measuresanti-PnPS antibodies to the additional serotypes (15B, 22F, 33F). Eachassay contains a combination of 13 or 7 spectrally distinct magneticmicrospheres coupled to PnPS conjugates (PnPS-PLL conjugates: PnPSconjugated to poly-L-Lysine).

Briefly, reference standard, controls and test sera were firstpre-adsorbed with two Pn absorbents; CWPS1 (cell wall polysaccharidefrom PnA containing C-polysaccharide) and CWPS2 (CWP from acapsular S.pneumoniae serotype 2) to block non-specific antibodies from binding tothe PnPS coating antigen. Following preadsorption, the PnPS-coupledmicrospheres were incubated with appropriately diluted referencestandard serum, controls or rabbit test sera. After incubation, eachmixture was washed and an R-Phycoerythrin-conjugated goat anti-rabbitIgG secondary antibody was added. Fluorescent signals (expressed asmedian fluorescence intensities (MFIs)) were measured using a Bio-Plexreader and correlated to the amount of bound PnPS-specific IgG. Valuesfor test sera are reported as (Units/mL, U/mL).

Serotype-specific OPAs were performed as described above. The OPA titeris the reciprocal of the highest serum dilution resulting in 50%reduction in the number of bacterial colony forming units (CFUs) whencompared to the control without serum (defined as the background CFU).The titer is interpolated from the two dilutions that encompass this 50%killing cut-off.

Rabbits immunized with the 20vPnC also demonstrated significantincreases in total IgG and functional OPA antibody titers againstserotypes common to the 16 v and 20 v formulations as well as to theadditional four serotypes (8, 10A, 11A, and 12F) (Table 22). A 2-logincrease in serum IgG levels across the 20 serotypes was inducedfollowing two immunizations. OPA GMTs elicited with the vaccine were atleast 27-fold above baseline. Low level OPA titers in pre-immune serafor serotypes 14 and 33F were similarly observed following 20vPnCvaccination, but again did not alter the robustness of thepost-vaccination antibody responses.

The 16vPnC and 20vPnC formulations elicited a robust humoral responsethat was both specific for Pneumococcal polysaccharides and associatedwith functional killing of the bacterium (see Tables 21 and 22). Inconclusion, studies shown in Examples 17 and 18 demonstrated goodimmunogenicity of both the 16vPnC and 20vPnC formulations.

TABLE 22 20vPnC Total IgG Concentrations and OPA Titers Total IgG (PndLIA) Opsonophagocytic Antibody (OPA) Wk 0 Wk 4 IgG GMC Wk Wk Wk 4 OPAGMT GMC GMC Wk 4 95% Ratio 0 4 95% CI Ratio Serotype (μg/ml) (μg/ml) CI(LCI-UCI) Wk4:Wk0 GMT GMT (LCI-UCI) Wk 4:Wk 0  1 0.08 28 19-43 379 4 106 69-164 27  3 0.08 116  76-176 1542 4 286 193-425 72  4 0.08 62 39-97821 4 1477  954-2287 369  5 0.08 49 33-71 648 4 509 350-742 127  6A 0.0330 14-66 1209 4 3682 2743-4944 849  6B 0.08 58 36-94 775 4 44693002-6653 1117  7F 0.02 62  39-101 3681 6 3226 2226-4675 500  9V 0.05 3019-48 644 6 956  634-1442 150 14 0.08 34 20-60 457 12 506 348-736 42 18C0.05 106  67-166 2115 4 1942 1263-2986 485 19A 0.08 112  73-171 1493 41580 1071-2332 395 19F 0.08 178 119-266 2372 4 3392 2085-5519 848 23F0.05 48  23-103 960 4 1514  889-2577 378 15B 0.05 70 51-98 1410 6 1332 949-1869 210 22F 0.10 172 118-250 1811 5 1304 1000-1700 279 33F 0.12 1410-20 120 54 1490 1117-1989 28  8 0.13 144 100-207 1149 4 1388  988-1949333 10A 0.13 54 31-94 433 5 1129  732-1741 236 11A 0.13 178 125-254 14237 10483  6373-17241 1434 12F 0.08 31 15-63 408 4 828  608-1127 191Abbreviations: GMC, geometric mean concentration; CI, confidenceinterval; LCI, lower confidence interval; UCI, upper confidenceinterval.

Example 19 Evaluation of Cross-Reactive Opsonophagocytic ImmuneResponses within Serogroup 9 of Streptococcus pneumoniae

The pneumococcal opsonophagocytic assay (OPA), which measures killing ofS. pneumoniae cells by phagocytic effector cells in the presence offunctional antibody and complement, is considered to be an importantsurrogate for evaluating the effectiveness of pneumococcal vaccines.

Materials and Methods

Two randomly selected subsets of immune sera from adults vaccinated witha 13-valent pneumococcal conjugate vaccine (13 v PnC) were tested in OPAassays for the serotypes 9V, 9A, 9L and 9N. The sera were collected fromU.S. clinical trials 6115A1-004 (N=59, post-vaccinated) and 6115A1-3005(N=66, matched pre- and post-vaccination), respectively.

Study 6115A1-3005 (ClinicalTrials.gov Identifier: NCT00546572) was aphase 3, randomized, active-controlled, modified double-blind trialevaluating the safety, tolerability, and immunogenicity of PREVNAR 13®compared with a 23-valent pneumococcal polysaccharide vaccine (23vPS) inambulatory elderly individuals aged 70 years and older who received 1dose of 23vPS at least 5 years before study enrollment (see:http://clinicaltrials.gov/ct2/show/NCT00546572; accessed on Mar. 31,2014).

Study 6115A1-004 (ClinicalTrials.gov Identifier: NCT00427895) was aphase 3, randomized, active-controlled, modified double-blind trialevaluating the safety, tolerability, and immunogenicity of a 13-valentpneumococcal conjugate vaccine (13vPnC) compared to a 23-valentpneumococcal polysaccharide vaccine (23vPS) in adults 60 to 64 years oldwho are naive to 23vPS and the safety, tolerability, and immunogenicityof 13vPnC in adults 18 to 59 years old who are naïve to 23vPS (see:http://clinicaltrials.gov/show/NCT00427895; accessed on Mar. 31, 2014).

The 13-valent pneumococcal conjugate vaccine (13vPnC) tested in thesestudies contained conjugates from pneumococcal serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, individually conjugated todiphtheria cross-reacting material 197 (CRM₁₉₇) carrier protein.

OPAs are used to measure functional antibodies in human sera against S.pneumoniae serotypes 9V, 9N, 9A and/or 9L. Test serum is set up in assayreactions that measure the ability of capsular polysaccharide specificimmunoglobulin to opsonize bacteria, trigger complement deposition,thereby facilitating phagocytosis and killing of bacteria by phagocytes.The OPA titer is defined as the reciprocal dilution that results in a50% reduction in bacterial count over control wells without test serum.The OPA titer is interpolated from the two dilutions that encompass this50% killing cut-off.

OPA procedures were based on methods described in Hu et al. (2005) ClinDiagn Lab Immunol122):287-295. Test heat-inactivated serum was seriallydiluted 2.5-fold and was added together with the target bacteria inassay plates and incubated for 30 minutes with shaking. DifferentiatedHL-60 cells (phagocytes) and baby rabbit serum (3- to 4-week old,PEL-FREEZ®, Arkansas, 12.5% final concentration) were then added to thewells, at an approximate effector to target ratio of 200:1, andincubated at 37° C. with shaking. To terminate the reaction, 80 μL of0.9% NaCl was added to all wells, mixed, and a 10 μL aliquot weretransferred to the wells of MULTISCREEN® HTS HV filter plates(MILLIPORE®) containing 200 μL of water. Liquid was filtered through theplates under vacuum, and 150 μL of HYSOY® medium was added to each welland filtered through. The filter plates were then incubated at 37° C.,5% CO₂ overnight and were then fixed with Destain Solution (Bio-RadLaboratories, Inc., Hercules, Calif.). The plates were then stained withCoomassie Blue and destained once. Colonies were imaged and enumeratedon a Cellular Technology Limited (CTL) (Shaker Heights, Ohio)IMMUNOSPOT® Analyzer.

Statistical Analysis: Pearson two-tailed correlations were calculated.

Results—OPA Responses in 9V, 9A, 9L and 9N

The cross-functional response from immune sera of adults immunized with13vPnC against serotypes 9A, 9L, and 9N, was evaluated in the respectivemicrocolony Opsonophagocytic Assays (mcOPAs), along with the homologousfunctional response to serotype 9V. Two randomly selected subsets ofimmune sera from adults vaccinated with 13vPnC were tested. The serawere collected from U.S. clinical trials 6115A1-004 (N=59,post-vaccinated) and 6115A1-3005 (N=66, matched pre- andpost-vaccination), respectively.

Subjects in study 6115A1-004 were previously naïve to any pneumococcalvaccination and received a single dose of 13vPnC as part of the studyprotocol. The immune sera from study 6115A1-004 shows a similarpercentage of responders for all the serogroups with values of 98.3%,98.3%, 100% and 93.2% for 9V, 9A, 9L and 9N respectively (FIG. 11),supporting the results from 6115A1-3005 (FIG. 12). A relative good OPAtiter correlations were observed between serotypes 9V and 9A (Pearsoncorrelation ρ=0.5456, p<0.0001) or 9L (ρ=0.7353, p<0.0001), but not with9N (ρ=0.1217, p<0.3627).

Subjects in study 6115A1-3005 had previously received 1 dose of 23vPS atleast 5 years before study enrollment and received a single dose of13vPnC as part of the study protocol. Matched pre- and post-vaccinationserum panel (N=66) from adults immunized with 13vPnC (study 6115A1-3005)was evaluated on OPA for the homologous response to serotype 9V and forcross-reactivity of anti-9V antibodies to serotypes 9A, 9L, and 9N. Asshown in FIG. 12, a relatively high immunity (percentage responders) to9V (84%), 9A (66%), 9L (82%) and 9N (86%) was detected in the OPA assaylikely due to their previous immunization with 23vPS, which includesunconjugated polysaccharides from serotypes 9V and 9N. However, thepercentage responders increased to 95% or more for all four serotypesafter vaccination with 13vPnC, which only contains serotype 9V conjugatefrom serogroup 9. The fold-rise in titer values are shown in Table 23and are similar between the serotypes also suggesting cross-reactivity.

TABLE 23 OPA Titer Fold-Rise Matched Pre- and Post-Vaccination, 13vPnCOPA Titers 9V 9A 9L 9N Pre Post Pre Post Pre Post Pre Post GMT 221 132341 308 165 706 322 693 Fold-rise 5.9 7.5 4.2 2.1

A more comprehensive analysis of the OPA titer distribution is shown inthe reverse cumulative distribution curves (RCDC) in FIGS. 13-16. TheRCDCs show an increase in serotype-specific immune response postvaccination for serotypes 9V, 9A, 9L and to a lesser extent 9N. Thecorrelation of the fold-rise of titer of individual matched/samplesbetween 9V 9A, 9V/9L, and 9V/9N were also analyzed using Pearson'scorrelation. Relatively good correlations of fold-rises of titers wereobserved between serotypes 9V and 9A (Pearson correlation ρ=0.8720,p<0.0001) or 9N (ρ=0.5801, p<0.0001), but to a lesser extent with 9L(ρ=0.1804, p<0.1640).

CONCLUSION

Based on these data, the 13vPnC vaccine is likely to provide broaderserotype coverage by providing additional protection against serotypes9A, 9L, and 9N.

Example 20 Cross-Functional OPA Responses Between Serotype 15B andSerotype 15C

Pneumococcal serogroup 15 includes four structurally-related serotypes:15A, 15B, 15C, and 15F. Serotypes 15B and 15C are undistinguishable bygenetic typing techniques and have similar capsular polysaccharide (PS)composition, except that the 15B-PS is the O-acetylated variant of15C-PS. To understand whether anti-capsular PS antibodies for serotype15B are functionally cross-reacting with serotype 15C, 10 rabbits wereimmunized with 16vPnC (see example 15) and 20vPnC (see example 16)vaccines both containing an immunogenic conjugate comprising S.pneumoniae serotype 15B capsular polysaccharide covalently linked toCRM₁₉₇ as disclosed herein as part of their formulation. Sera from pre-and post-vaccination were tested in OPA assays against serotypes 15B and15C target pneumococcal strains.

Of the 10 rabbits from each group, 100% had OPA response to serotype 15Bfollowing immunization with a serotype 15B conjugate. Of these samesamples, 100% had OPA response to serotype 15C as well (Table 24 andTable 25). Low OPA titers were observed in prevaccination sera in 15COPA. However, over 10-fold GMT OPA titer increase with post vaccinationsera compared to pre vaccination demonstrated that the immunogenicconjugates of the invention induces the formation of antibodies capableof killing serotype 15B and 15C Streptococcus pneumonia in an OPA.

TABLE 24 OPA Titers Against serotypes 15B and 15C strains in Rabbit SeraPre and Post vaccination with 16vPnC 15B OPA 15C OPA Animal wk 0 wk 4 wk0 wk 4 1 4 4129 50 2524 2 4 1645 182 472 3 4 1131 126 818 4 4 3199 501189 5 4 2664 36 727 6 4 4589 68 2492 7 11 3601 169 1137 8 4 1838 165672 9 4 1334 98 528 10  4 1108 204 2425 GMT 4 2222 98 1075

TABLE 25 OPA Titers Against serotypes 15B and 15C strains in Rabbit SeraPre and Post vaccination with 20vPnC 15B OPA 15C OPA Animal wk 0 wk 4 wk0 wk 4 1 4 3784 indeterminable* 2353 2 4 862 480 938 3 4 3056  69 1497 44 1948 indeterminable* 1316 5 4 2360  4 4665 6 4 1594 indeterminable*1835 7 4 4943 172 4085 8 4 2419 117 1458 9 4 1245 indeterminable* 52710  4 616 indeterminable* 545 GMT 4 1917  77 1515 *Titer cannot bedetermined due to bad killing curves

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are herebyincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

The invention claimed is:
 1. An immunogenic composition comprising aStreptococcus pneumoniae serotype 15B capsular polysaccharide-carrierprotein glycoconjugate wherein: (a) said serotype 15B capsularpolysaccharide-carrier protein glycoconjugate has a molecular weight ofbetween 1000 kDa and 10,000 kDa; (b) the degree of conjugation of saidserotype 15B capsular polysaccharide-carrier protein glycoconjugate isbetween 2 and 15; (c) the ratio (w/w) of serotype 15B capsularpolysaccharide to the carrier protein in said serotype 15B capsularpolysaccharide-carrier protein glycoconjugate is between 0.5 and 3; (d)said carrier protein is CRM₁₉₇, and wherein said immunogenic compositionfurther comprises at least one capsular polysaccharide-carrier proteinglycoconjugate comprising a S. pneumoniae serotype selected fromserotype 8, 10A, 11A, 12F and 33F.
 2. The immunogenic composition ofclaim 1, further comprising a capsular polysaccharide-carrier proteinglycoconjugate from S. pneumoniae serotype 33F.
 3. The immunogeniccomposition of claim 1, further comprising a capsularpolysaccharide-carrier protein glycoconjugate from S. pneumoniaeserotype 12F.
 4. The immunogenic composition of claim 1, furthercomprising a capsular polysaccharide-carrier protein glycoconjugate fromS. pneumoniae serotype 10A.
 5. The immunogenic composition of claim 1,further comprising a capsular polysaccharide-carrier proteinglycoconjugate from S. pneumoniae serotype 11A.
 6. The immunogeniccomposition of claim 1, further comprising a capsularpolysaccharide-carrier protein glycoconjugate from S. pneumoniaeserotype
 8. 7. The immunogenic composition of claim 1, furthercomprising capsular polysaccharide-carrier protein glycoconjugates fromS. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19Fand 23F.
 8. The immunogenic composition of claim 1, wherein theimmunogenic composition is an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20-valent pneumococcal capsular polysaccharide-carrier proteinglycoconjugate composition.
 9. The immunogenic composition of claim 1,wherein said serotype 15B capsular polysaccharide-carrier proteinglycoconjugate comprises less than about 50% of free serotype 15Bcapsular polysaccharide compared to the total amount of serotype 15Bcapsular polysaccharide.
 10. The immunogenic composition of claim 1,wherein said serotype 15B capsular polysaccharide-carrier proteinglycoconjugate comprises at least 0.1 mM acetate per mM serotype 15Bcapsular polysaccharide.
 11. The immunogenic composition of claim 1,wherein the ratio of mM acetate per mM serotype 15B capsularpolysaccharide in the serotype 15B capsular polysaccharide-carrierprotein glycoconjugate to mM acetate per mM serotype 15B capsularpolysaccharide in the isolated polysaccharide is at least 0.6.
 12. Theimmunogenic composition of claim 1, wherein the ratio of mM acetate permM serotype 15B capsular polysaccharide in the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate to mM acetate per mMserotype 15B capsular polysaccharide in the activated polysaccharide isat least 0.6.
 13. The immunogenic composition of claim 1, wherein saidserotype 15B capsular polysaccharide-carrier protein glycoconjugatecomprises at least 0.1 mM glycerol per mM serotype 15B capsularpolysaccharide.
 14. The immunogenic composition of claim 1, wherein saidserotype 15B capsular polysaccharide-carrier protein glycoconjugatecomprises a saccharide having a molecular weight of between 10 kDa and1,500 kDa.
 15. The immunogenic composition of claim 1, wherein saidserotype 15B capsular polysaccharide-carrier protein glycoconjugate isprepared using reductive amination.
 16. The immunogenic composition ofclaim 1, wherein each dose of said immunogenic composition comprises 0.1to 100 μg of polysaccharide of the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate.
 17. The immunogeniccomposition of claim 1, wherein each dose of said immunogeniccomposition comprises 10 μg to 150 μg of carrier protein of the serotype15B capsular polysaccharide-carrier protein glycoconjugate.
 18. Theimmunogenic composition of claim 1, wherein said immunogenic compositionfurther comprises an antigen from other pathogens.
 19. The immunogeniccomposition of claim 1, wherein said immunogenic composition furthercomprises a conjugated N. meningitides serogroup A capsular saccharide(MenA), a conjugated N. meningitides serogroup W135 capsular saccharide(MenW135), a conjugated N. meningitides serogroup Y capsular saccharide(MenY), a conjugated N. meningitides serogroup C capsular saccharide(MenC), or a combination thereof.
 20. The immunogenic composition ofclaim 1, wherein said immunogenic composition further comprises at leastone adjuvant.
 21. The immunogenic composition of claim 20, wherein saidat least one adjuvant is selected from the group consisting of aluminum,calcium phosphate, a liposome, an oil-in-water emulsion, andpoly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.22. The immunogenic composition of claim 21, wherein said adjuvant is analuminum adjuvant selected from the group consisting of aluminumphosphate, aluminum sulfate and aluminum hydroxide.
 23. The immunogeniccomposition of claim 1, wherein the degree of conjugation of theserotype 15B capsular polysaccharide-carrier protein glycoconjugate isbetween 3 and
 5. 24. The immunogenic composition of claim 1, wherein theserotype 15B capsular polysaccharide-carrier protein glycoconjugate hasa molecular weight of between 4,000 kDa and 10,000 kDa.
 25. Theimmunogenic composition of claim 1, wherein the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate has a molecular weight ofbetween 4,000 kDa and 8,000 kDa.
 26. The immunogenic composition ofclaim 1, wherein the ratio (w/w) of serotype 15B capsular polysaccharideto CRM₁₉₇ carrier protein in said serotype 15B capsularpolysaccharide-carrier protein glycoconjugate is between 0.5 and 1.5.27. The immunogenic composition of claim 1, wherein the ratio (w/w) ofserotype 15B capsular polysaccharide to CRM₁₉₇ carrier protein in saidserotype 15B capsular polysaccharide-carrier protein glycoconjugate isbetween 0.7 and 1.2.
 28. The immunogenic composition of claim 1, whereinthe 15B capsular polysaccharide-carrier protein glycoconjugate comprisesat least 0.7 mM acetate per mM serotype 15B capsular polysaccharide. 29.The immunogenic composition of claim 1, wherein less than 15% of theserotype 15B capsular polysaccharide in the composition is free serotype15B capsular polysaccharide.
 30. The immunogenic composition of claim 1,wherein the isolated serotype 15B capsular polysaccharide used togenerate the serotype 15B capsular glycoconjugate is between 150-300kDa.
 31. The immunogenic composition of claim 1, wherein the serotype15B capsular polysaccharide used to generate the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate is chemically activated tomake the polysaccharide capable of reacting with the CRM₁₉₇ carrierprotein, and wherein the activated serotype 15B capsular polysaccharideis between 100-250 kDa.
 32. The immunogenic composition of claim 1,wherein the serotype 15B capsular polysaccharide was conjugated to theCRM₁₉₇ carrier in dimethyl sulfoxide (DMSO).
 33. The immunogeniccomposition of claim 1, wherein at least 99% of the serotype 15Bcapsular polysaccharide in the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate has retained O-acetylationas compared to the input serotype 15B capsular polysaccharide used togenerate the serotype 15B capsular polysaccharide-carrier proteinglycoconjugate.
 34. The immunogenic composition of claim 1, wherein thedegree of conjugation of the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate is between 3 and 5;wherein the serotype 15B capsular polysaccharide-carrier proteinglycoconjugate has a molecular weight of between 4,000 kDa and 10,000kDa; wherein the ratio (w/w) of serotype 15B capsular polysaccharide toCRM₁₉₇ carrier protein in said serotype 15B capsularpolysaccharide-carrier protein glycoconjugate is between 0.5 and 1.5;wherein the serotype 15B capsular polysaccharide-carrier proteinglycoconjugate comprises at least 0.7 mM acetate per mM serotype 15Bcapsular polysaccharide; and wherein less than 10% of the serotype 15Bcapsular polysaccharide in the composition is free serotype 15B capsularpolysaccharide.
 35. The immunogenic composition of claim 1, wherein thedegree of conjugation of the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate is between 3 and 5;wherein the serotype 15B capsular polysaccharide-carrier proteinglycoconjugate has a molecular weight of between 4,000 kDa and 8,000kDa; wherein the ratio (w/w) of serotype 15B capsular polysaccharide toCRM₁₉₇ carrier protein in said serotype 15B capsularpolysaccharide-carrier protein glycoconjugate is between 0.7 and 1.2;wherein the serotype 15B capsular polysaccharide-carrier proteinglycoconjugate comprises at least 0.7 mM acetate per mM serotype 15Bcapsular polysaccharide; and wherein less than 10% of the serotype 15Bcapsular polysaccharide in the composition is free serotype 15B capsularpolysaccharide.
 36. The immunogenic composition of claim 1, wherein theserotype 15B capsular polysaccharide used to generate the serotype 15Bcapsular polysaccharide-carrier protein glycoconjugate is chemicallyactivated to make the polysaccharide capable of reacting with the CRM₁₉₇carrier protein; wherein the activated serotype 15B capsularpolysaccharide is between 100-250 kDa; and wherein the serotype 15Bcapsular polysaccharide was conjugated to the CRM₁₉₇ carrier protein indimethyl sulfoxide (DMSO).
 37. The immunogenic composition of claim 33,wherein the serotype 15B capsular polysaccharide used to generate theserotype 15B capsular polysaccharide-carrier protein glycoconjugate ischemically activated to make the polysaccharide capable of reacting withthe CRM₁₉₇ carrier protein; wherein the activated serotype 15B capsularpolysaccharide is between 100-250 kDa; and wherein the serotype 15Bcapsular polysaccharide was conjugated to the CRM₁₉₇ carrier protein indimethyl sulfoxide (DMSO).
 38. The immunogenic composition of claim 34,wherein the serotype 15B capsular polysaccharide used to generate theserotype 15B capsular polysaccharide-carrier protein glycoconjugate ischemically activated to make the polysaccharide capable of reacting withthe CRM₁₉₇ carrier protein; wherein the activated serotype 15B capsularpolysaccharide is between 100-250 kDa; and wherein the serotype 15Bcapsular polysaccharide was conjugated to the CRM₁₉₇ carrier protein indimethyl sulfoxide (DMSO).
 39. The immunogenic composition of claim 36,wherein at least 99% of the serotype 15B capsular polysaccharide in theserotype 15B capsular polysaccharide-carrier protein glycoconjugate hasretained O-acetylation as compared to the input serotype 15B capsularpolysaccharide used to generate the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate.
 40. The immunogeniccomposition of claim 37, wherein at least 99% of the serotype 15Bcapsular polysaccharide in the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate has retained O-acetylationas compared to the input serotype 15B capsular polysaccharide used togenerate the serotype 15B capsular polysaccharide-carrier proteinglycoconjugate.
 41. The immunogenic composition of claim 38, wherein atleast 99% of the serotype 15B capsular polysaccharide in the serotype15B capsular polysaccharide-carrier protein glycoconjugate has retainedO-acetylation as compared to the input serotype 15B capsularpolysaccharide used to generate the serotype 15B capsularpolysaccharide-carrier protein glycoconjugate.
 42. The immunogeniccomposition of claim 1, further comprising capsularpolysaccharide-carrier protein glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A,19F, 22F, 23F, and 33F; wherein each of the glycoconjugates comprisesCRM₁₉₇ carrier protein.
 43. The immunogenic composition of claim 33,further comprising capsular polysaccharide-carrier proteinglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8,9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F; wherein each ofthe glycoconjugates comprises CRM₁₉₇ carrier protein.
 44. Theimmunogenic composition of claim 34, further comprising capsularpolysaccharide-carrier protein glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A,19F, 22F, 23F, and 33F; wherein each of the glycoconjugates comprisesCRM₁₉₇ carrier protein.
 45. The immunogenic composition of claim 35,further comprising capsular polysaccharide-carrier proteinglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8,9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F; wherein each ofthe glycoconjugates comprises CRM₁₉₇ carrier protein.
 46. Theimmunogenic composition of claim 36, further comprising capsularpolysaccharide-carrier protein glycoconjugates from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A,19F, 22F, 23F, and 33F; wherein each of the glycoconjugates comprisesCRM₁₉₇ carrier protein.
 47. The immunogenic composition of claim 37,further comprising capsular polysaccharide-carrier proteinglycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8,9V, 10A, 11A, 12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F; wherein each ofthe glycoconjugates comprises CRM₁₉₇ carrier protein.
 48. Theimmunogenic composition of claim 18, wherein said antigen is selectedfrom the group consisting of a diphtheria toxoid (D), a tetanus toxoid(T), a pertussis antigen (P), an acellular pertussis antigen (Pa), ahepatitis B virus (HBV) surface antigen (HBsAg), a hepatitis A virus(HAV) antigen, a conjugated Haemophilus influenzae type b capsularsaccharide (Hib), and an inactivated poliovirus vaccine (IPV) antigen.